U.S. patent application number 09/823356 was filed with the patent office on 2001-09-27 for human membrane-spanning proteins.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Bandman, Olga, Baughn, Mariah R., Corley, Neil C., Guegler, Karl J., Hillman, Jennifer L., Kaser, Matthew R., Lal, Preeti, Shah, Purvi, Tang, Y. Tom, Yue, Henry.
Application Number | 20010025098 09/823356 |
Document ID | / |
Family ID | 21904776 |
Filed Date | 2001-09-27 |
United States Patent
Application |
20010025098 |
Kind Code |
A1 |
Tang, Y. Tom ; et
al. |
September 27, 2001 |
Human membrane-spanning proteins
Abstract
The invention provides a human membrane spanning proteins (MSPs)
and polynucleotides which identify and encode MSPs. The invention
also provides expression vectors, host cells, antibodies, agonists,
and antagonists. The invention also provides methods for treating
or preventing disorders associated with expression of MSPs.
Inventors: |
Tang, Y. Tom; (San Jose,
CA) ; Bandman, Olga; (Mountain View, CA) ;
Lal, Preeti; (Santa Clara, CA) ; Hillman, Jennifer
L.; (Mountain View, CA) ; Yue, Henry;
(Sunnyvale, CA) ; Corley, Neil C.; (Mountain View,
CA) ; Guegler, Karl J.; (Menlo Park, CA) ;
Kaser, Matthew R.; (Castro Valley, CA) ; Baughn,
Mariah R.; (San leandro, CA) ; Shah, Purvi;
(Sunnyvale, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
21904776 |
Appl. No.: |
09/823356 |
Filed: |
March 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09823356 |
Mar 30, 2001 |
|
|
|
09039307 |
Mar 13, 1998 |
|
|
|
Current U.S.
Class: |
530/350 ;
435/325; 435/6.14; 435/69.1; 530/388.22; 536/23.5 |
Current CPC
Class: |
C07K 14/705
20130101 |
Class at
Publication: |
530/350 ; 435/6;
435/69.1; 435/325; 536/23.5; 530/388.22 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/02; C07K 014/705; C07K 016/18 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:1-17, b) a naturally occurring
polypeptide comprising an amino acid sequence at least 90%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-17, c) a biologically active fragment of
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-17.
2. An isolated polypeptide of claim 1 selected from the group
consisting of SEQ ID NO:1-17.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 selected from the group
consisting of SEQ ID NO:18-34.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method for producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. An isolated antibody which specifically binds to a polypeptide
of claim 1.
11. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:18-34, b) a
naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90% identical to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:18-34, c) a
polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to the polynucleotide of b), and e) an
RNA equivalent of a)-d).
12. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 11.
13. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
14. A method of claim 13, wherein the probe comprises at least 60
contiguous nucleotides.
15. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
16. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
17. A composition of claim 16, wherein the polypeptide has an amino
acid sequence selected from the group consisting of SEQ ID
NO:1-17.
18. A method for treating a disease or condition associated with
decreased expression of functional MSP, comprising administering to
a patient in need of such treatment the composition of claim
16.
19. A method for screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
20. A composition comprising an agonist compound identified by a
method of claim 19 and a pharmaceutically acceptable excipient.
21. A method for treating a disease or condition associated with
decreased expression of functional MSP, comprising administering to
a patient in need of such treatment a composition of claim 20.
22. A method for screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
23. A composition comprising an antagonist compound identified by a
method of claim 22 and a pharmaceutically acceptable excipient.
24. A method for treating a disease or condition associated with
overexpression of functional MSP, comprising administering to a
patient in need of such treatment a composition of claim 23.
25. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, said method comprising the steps of: a)
combining the polypeptide of claim 1 with at least one test
compound under suitable conditions, and b) detecting binding of the
polypeptide of claim 1 to the test compound, thereby identifying a
compound that specifically binds to the polypeptide of claim 1.
26. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
27. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
28. A method for assessing toxicity of a test compound, said method
comprising: a) treating a biological sample containing nucleic
acids with the test compound; b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 11 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 11 or fragment thereof; c)
quantifying the amount of hybridization complex; and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
29. A diagnostic test for a condition or disease associated with
the expression of MSP in a biological sample comprising the steps
of: a) combining the biological sample with an antibody of claim
10, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex; and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
30. The antibody of claim 10, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
31. A composition comprising an antibody of claim 10 and an
acceptable excipient.
32. A method of diagnosing a condition or disease associated with
the expression of MSP in a subject, comprising administering to
said subject an effective amount of the composition of claim
31.
33. A composition of claim 31, wherein the antibody is labeled.
34. A method of diagnosing a condition or disease associated with
the expression of MSP in a subject, comprising administering to
said subject an effective amount of the composition of claim
33.
35. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 10 comprising: a) immunizing
an animal with a polypeptide having an amino acid sequence selected
from the group consisting of SEQ ID NO:1-17, or an immunogenic
fragment thereof, under conditions to elicit an antibody response;
b) isolating antibodies from said animal; and c) screening the
isolated antibodies with the polypeptide, thereby identifying a
polyclonal antibody which binds specifically to a polypeptide
having an amino acid sequence selected from the group consisting of
SEQ ID NO:1-17.
36. An antibody produced by a method of claim 35.
37. A composition comprising the antibody of claim 36 and a
suitable carrier.
38. A method of making a monoclonal antibody with the specificity
of the antibody of claim 10 comprising: a) immunizing an animal
with a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID NO:1-17, or an immunogenic fragment
thereof, under conditions to elicit an antibody response; b)
isolating antibody producing cells from the animal; c) fusing the
antibody producing cells with immortalized cells to form monoclonal
antibody-producing hybridoma cells; d) culturing the hybridoma
cells; and e) isolating from the culture monoclonal antibody which
binds specifically to a polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO:1-17.
39. A monoclonal antibody produced by a method of claim 38.
40. A composition comprising the antibody of claim 39 and a
suitable carrier.
41. The antibody of claim 10, wherein the antibody is produced by
screening a Fab expression library.
42. The antibody of claim 10, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
43. A method for detecting a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-17 in a
sample, comprising the steps of: a) incubating the antibody of
claim 10 with a sample under conditions to allow specific binding
of the antibody and the polypeptide; and b) detecting specific
binding, wherein specific binding indicates the presence of a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-17 in the sample.
44. A method of purifying a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-17 from
a sample, the method comprising: a) incubating the antibody of
claim 10 with a sample under conditions to allow specific binding
of the antibody and the polypeptide; and b) separating the antibody
from the sample and obtaining the purified polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-17.
45. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:1.
46. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:2.
47. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:3.
48. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:4.
49. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:5.
50. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:6.
51. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:7.
52. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:8.
53. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:9.
54. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:10.
55. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:11.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:12.
57. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:13.
58. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:14.
59. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:15.
60. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:16.
61. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:17.
62. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:18.
63. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:19.
64. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:20.
65. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:21.
66. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:22.
67. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:23.
68. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:24.
69. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:25.
70. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:26.
71. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:27.
72. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:28.
73. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:29.
74. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:30.
75. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:31.
76. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:32.
77. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:33.
78. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:34.
Description
[0001] This application is a CONTINUATION application of U.S.
application Ser. No. 09/039,307, filed on Mar. 13, 1998, originally
entitled HUMAN MEMBRANE SPANNING PROTEINS, which is hereby
expressly incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of human membrane spanning proteins and to the use of
these sequences in the diagnosis, treatment, and prevention of
neoplastic, immunological, and reproductive disorders.
BACKGROUND OF THE INVENTION
[0003] Eukaryotic organisms are distinct from prokaryotes in
possessing many intracellular organelle and vesicle structures.
Many of the metabolic reactions which distinguish eukaryotic
biochemistry from prokaryotic biochemistry take place within these
structures. In particular, many cellular functions require very
strict reagent conditions, and the organelles and vesicles enable
compartmentalization and isolation of reactions which might
otherwise cripple cytosolic metabolic processes. The organelles are
bounded by single or double membranes each made up of lipid bilayer
sheets and include mitochondria; smooth and rough endoplasmic
reticuli; sarcoplasmic reticulum; and the Golgi body. The lipid
bilayer sheets are composed of phosphoglycerides, fatty acids,
cholesterol, glycolipids, proteoglycans, and proteins. The vesicles
are bounded by single membranes and include phagosomes; lysosomes;
endosomes; peroxisomes; and secretory vesicles.
[0004] Biological membranes are highly selective permeability
barriers that contain ion pumps, gates, and specific receptors for
external stimuli which transmit biochemical signals across the
membranes. These membranes also contain proteins which interact
with these pumps, gates, and receptors to amplify and regulate
transmision of these signals.
[0005] Plasma Membrane Proteins
[0006] Plasma membrane proteins (MPs) are divided into two groups
based upon the mechanism of protein removal from the membrane.
Extrinsic or peripheral membrane proteins can be removed using
extremes of ionic strength or pH, urea, or other disruptors of
protein interactions. Intrinsic or integral membrane proteins are
released only when the lipid bilayer of the membrane is dissolved
by detergent.
[0007] The majority of known integral membrane proteins are
transmembrane proteins which are characterized by an extracellular,
a transmembrane, and an intracellular domain. Transmembrane
proteins are typically embedded in the cell membrane by one or more
regions comprising 15 to 25 hydrophobic amino acids which are
predicted to adopt an .alpha.-helical conformation. Transmembrane
proteins are classified as bitopic (Types I and II) and polytopic
(Types III and IV) (Singer, S. J. (1990) Annu. Rev. Cell Biol.
6:247-296). Bitopic proteins span the membrane once while polytopic
proteins contain multiple membrane-spanning segments. Type III
integral membrane proteins have multiple transmembrane stretches of
hydrophobic residues. Transmembrane proteins carry out a variety of
important cellular functions such as acting as cell-surface
receptor proteins involved in signal transduction, e.g., growth
factor and differentiation factor receptors and
receptor-interacting proteins; transport of ions or metabolites,
e.g., gap junction channels (connexins), and ion channels; cell
anchoring proteins, extracellular matrix (ECM)-binding proteins,
lectins, integrins, and fibronectins; or cell recognition
molecules, e.g., cluster of differentiation (CD) antigens,
glycoproteins and mucins.
[0008] Many MPs contain amino acid sequence motifs that serve to
localize proteins to specific subcellular sites. Examples of these
motifs include, e.g., PDZ domains, KDEL, RGD, NGR, GSL, von
Willebrand factor A (vWFA) domains, and EGF-like domains.
Furthermore, MPs may also contain amino acid sequence motifs that
serve to interact with extracellular or intracellular molecules,
such as carbohydrate recognition domains (CRD). RGD, NGR, GSL
motif-containing peptides have been used as drug delivery agents in
targeted cancer treatment of tumor vasculature (Arap, W. et al.
(1998) Science 279:377-380).
[0009] Chemical modification of amino acid residue side chains
alters the manner in which a protein interacts with other
molecules, for example, phospholipid membranes. Examples of such
chemical modifications to amino acid residue side chains are
covalent bond formiation with glycosaminoglycans, oligosaccharides,
phospholipids, acetyl and palmitoyl moieties, ADP-ribose,
phosphate, and sulphate groups.
[0010] Premessenger RNA encoding membrane proteins may have
alternative splice sites which give rise to proteins encoded by the
same gene but with different messenger RNA and amino acid
sequences. Splice variant membrane proteins may interact with other
ligand and protein isoforms.
[0011] Tetraspan Family Proteins
[0012] The transmembrane 4 superfamily (TM4SF) or tetraspan family
are a multigene family encoding type III integral membrane proteins
(Wright, M. D. and Tomlinson, M. G. (1994) Immunol. Today 15:588).
The TM4SF family comprises a superfamily of membrane proteins which
traverse the cell membrane four times. Members of the TM4SF include
a number of platelet and endothelial cell membrane proteins, the
platelet and melanoma-associated antigens, leukocyte surface
glycoproteins, the colonal carcinoma antigens, the tumor-associated
antigens, and surface proteins of the schistosome parasites
(Jankowski, S. A. (1994) Oncogene 9:1205-1211). The members of the
TM4SF share about 25-30% amino acid sequence identity with one
another.
[0013] A number of TM4SF members have been implicated in signal
transduction, control of cell adhesion, regulation of cell growth
and proliferation, including development and oncogenesis, and
motility, including the ability to suppress metastatic potential.
Expression of a number of TM4SF proteins is associated with a
variety of tumors and the level of expression may be altered when
cells are growing or activated.
[0014] Proton Pumps
[0015] Proton ATPases are a large class of membrane-proteins that
use the energy of ATP hydrolysis to generate an electrochemical
proton gradient across a membrane. The resultant gradient may be
used to transport other ions across the membrane (Na.sup.+,
K.sup.+, or Cl.sup.-) or to maintain an acidic environment
important to the function of many cellular vesicles (Mellman, I. et
al. (1986) Annu. Rev. Biochem. 55:663-700). Proton ATPases are
further subdivided into the mitochondrial F-ATPases, the plasma
membrane ATPases, and the vacuolar ATPases. The vacuolar ATPases
establish and maintain an acidic pH within various vesicles
involved in the processes of endocytosis and exocytosis.
[0016] Proton-coupled, 12 membrane-spanning domain transporters
such as PEPT 1 and PEPT 2 are responsible for gastrointestinal
absorption and for renal reabsorbtion of peptides using an
electrochemical H.sup.+ gradient as the driving force. A
heterodimeric peptide transporter, consisting of TAP 1 and TAP 2,
is associated with antigen processing. Peptide antigens are
transported across the membrane of the endoplasmic reticulum so
they can be presented to the major histocompatibility complex class
I molecules. Each TAP protein consists of multiple hydrophobic
membrane spanning segments and a highly conserved ATP-binding
cassette (Boll, M. et al. (1996) Proc. Natl. Acad. Sci. USA
93:284-289). Pathogenic microorganisms, such as herpes simplex
virus, may encode inhibiotrs of TAP-mediated peptide transport in
order to evade immune surveillance (Marusina, K. and Manaco, J. J.
(1996) Curr. Opin. Hematol. 3:19-26).
[0017] Scavenger Receptors
[0018] Macrophage scavenger receptors with broad ligand specificity
have been suggested to take part in the binding of low density
lipoproteins (LDL) and foreign antigens. Scavenger receptors types
I and II are trimeric membrane proteins with a small N-terminal
intracellular domain, a transmembrane domain, a large extracellular
domain, and a C-terminal cysteine-rich domain. The extracellular
domain contains a short spacer domain, an .alpha.-helical
coiled-coil domain, and a triple helical collagenous domain. These
receptors have been shown to bind a spectrum of ligands, including
chemically modified lipoproteins and albumin, polyribonucleotides,
polysaccharides, phospholipids, and asbestos (Matsumoto, A. et al.
(1990) Proc. Natl. Acad. Sci. USA 87:9133-9137; and Elomaa, O. et
al. (1995) Cell 80:603-609).
[0019] The scavenger receptors are thought to play a key role in
atherogenesis by mediating uptake of modified LDL in arterial
walls, and in host defense by binding bacterial endotoxins,
bacteria, and protozoa.
[0020] Ion Channels
[0021] Ion channels are found in the plasma membranes of virtually
every cell in the body. For example, chloride channels mediate a
variety of cellular functions including regulation of membrane
potentials and absorption and secretion of ions across epithelial
membranes. When present in intracellular membranes of the Golgi
apparatus and endocytic vesicles, chloride channels also regulate
organelle pH (see, e.g., Greger, R. (1988) Annu. Rev. Physiol.
50:111-122). Electrophysiological and pharmacological measurements
including ion conductance, current-voltage relationships, and
sensitivity to modulators suggest that different chloride channels
exist in muscles, neurons, fibroblasts, epithelial cells, and
lymphocytes.
[0022] Many of the channels have sites for phosphorylation by one
or more protein kinases including protein kinase A, protein kinase
C, tyrosine kinase, and casein kinase II, all of which regulate ion
channel activity in cells. Inapproriate phosphorylation of proteins
in cells has been linked to changes in cell cycle events and
differentiation status. Changes in the cell cycle have been linked
to induction of apoptosis or cancer. Changes in differentiation
status of the cell have been linked to, for example, immune
diseases and disorders, diseases and disorders of skeletal muscle,
and diseases and disorders of the reproductive system.
[0023] G-Protein Coupled Receptors
[0024] G-protein coupled receptors (GPCR) are a superfamily of
integral membrane proteins which transduce extracellular signals.
GPCRs include receptors for biogenic amines; for lipid mediators of
inflammation, peptide hormones, and sensory signal mediators.
[0025] The structure of these highly-conserved receptors consists
of seven hydrophobic transmembrane (serpentine) regions, cysteine
disulfide bridges between the second and third extracellular loops,
an extracellular N-terminus, and a cytoplasmic C-terminus. Three
extracellular loops alternate with three intracellular loops to
link the seven transmembrane regions. The most conserved parts of
these proteins are the transmembrane regions and the first two
cytoplasmic loops. A conserved, acidic-Arg-aromatic residue triplet
present in the second cytoplasmic loop may interact with the G
proteins. The consensus pattern of the G-protein coupled receptors
signature (PS00237; SWISSPROT) is characteristic of most proteins
belonging to this superfamily (Watson, S. and S. Arkinstall (1994)
The G-protein Linked Receptor Facts Book, Academic Press, San Diego
Calif., pp. 2-6).
[0026] Mutations and changes in transcriptional activation of G
protein-encoding genes have been associated with, for example,
schizophrenia, Parkinson's disease, Alzheimer's disease, drug
addiction, and feeding disorders.
[0027] ABC Transporters
[0028] The ATP-binding cassette (ABC) transporters, also called the
"traffic ATPases", comprise a superfamily of membrane proteins that
mediate transport and channel functions in prokaryotes and
eukaryotes (Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8:67-113).
ABC proteins share a similar overall structure and significant
sequence homology. All ABC proteins contain a conserved domain of
approximately two hundred amino acid residues which includes one or
more nucleotide binding domains. Mutations in ABC transporter genes
are associated with, for example, hyperbilirubinemia
II/Dubin-Johnson syndrome, recessive Stargardt's disease, X-linked
adrenoluekodystrophy, multidrug resistance, celiac disease, and
cystic fibrosis.
[0029] Tumor Antigens
[0030] Tumor antigens are surface molecules that are differentially
expressed in tumor cells relative to non-tumor tissues. Tumor
antigens distinguish tumor cells immunologically from normal cells
and provide diagnostic and therapeutic targets for human cancers
(Takagi, S. et al. (1995) Int. J. Cancer 61:706-715; Liu, E. et al.
(1992) Oncogene 7:1027-1032).
[0031] Mitochondrial Membrane Proteins
[0032] The mitochondrial electron transport (or respiratory) chain
is a series of three enzyme complexes in the mitochondrial membrane
that is responsible for the transport of electrons from NADH to
oxygen and the coupling of this oxidation to the synthesis of ATP
(oxidative phosphorylation). ATP then provides the primary source
of energy for driving the many energy-requiring reactions of a
cell.
[0033] Most of the protein components of the mitochondrial
respiratory chain are the products of nuclear encoded genes that
are imported into the mitochondria, and the remainder are products
of mitochondrial genes. Defects and altered expression of enzymes
in the respiratory chain are associated with a variety of disease
conditions in man, including, for example, neurodegenerative
diseases, myopathies, and cancer.
[0034] Endopolasmic Reticulum Membrane Proteins
[0035] The normal functioning of the eukaryotic cell requires that
all newly synthesized proteins be correctly folded, modified, and
delivered to specific intra- and extracellular sites. Newly
synthesized membrane and secretory proteins enter a cellular
sorting and distribution network during or immediately after
synthesis (cotranslationally or posttranslationally) and are routed
to specific locations inside and outside of the cell. The initial
compartment in this process is the endoplasmic reticulum (ER) where
proteins undergo modifications such as glycosylation, disulfide
bond formation, and assembly into oligomers. The proteins are then
transported through an additional series of membrane-bound
compartments which include the various cisternae of the Golgi
complex, where further carbohydrate modifications occur. Transport
between compartments occurs by means of vesicles that bud and fuse
in a specific manner. Once within the secretory pathway, proteins
do not have to cross a membrane to reach the cell surface.
[0036] The majority of proteins processed through the ER are
transported out of the organelle however some are retained. The
signal for retention in the ER in mammalian cells consists of the
tetrapeptide sequence, KDEL, located at the carboxy terminus of
proteins (Munro, S. (1986) Cell 46:291-300). Proteins containing
this sequence leave the ER but are quickly retrieved from the early
Golgi compartment and returned to the ER, while proteins without
this signal continue through the distribution pathway.
[0037] Disruptions in the cellular secretory pathway have been
implicated in several human diseases. In familial
hypercholesterolemia the low density lipoprotein receptors remain
in the ER, rather than moving to the cell surface (Pathak, R. K.
(1988) J. Cell Biol. 106:1831-1841).
[0038] Presenilins are localized to the ER and may regulate
cellular calcium homeostasis in early-onset Alzheimer's disease and
in Down syndrome. Changes in ER-derived calcium homeostasis have
also been associated with, for example, cardiomyopathy, cardiac
hypertrophy, myotonic dystrophy, Brody disease, Smith-McCort
dysplasia, and diabetes mellitus.
[0039] Intercellular Communication Membrane Proteins
[0040] Intercellular communication is essential for the development
and survival of multicellular organisms. Cells communicate with one
another through the secretion and uptake of protein signaling
molecules. The uptake of proteins into the cell is achieved by
endocytosis, in which the interaction of signaling molecules with
the plasma membrane surface, often via binding to specific
receptors, results in the formation of plasma membrane-derived
vesicles that enclose and transport the molecules into the cytosol.
The secretion of proteins from the cell is achieved by exocytosis,
in which molecules inside of the cell are packaged into
membrane-bound transport vesicles derived from the trans Golgi
network. These vesicles fuse with the plasma membrane and release
their contents into the surrounding extracellular space.
Endocytosis and exocytosis result in the removal and addition of
plasma membrane components, and the recycling of these components
is essential to maintain the integrity, identity, and functionality
of both the plasma membrane and internal membrane-bound
compartments.
[0041] Endocytosis involves the internalizing of nutrients, solutes
or small particles (pinocytosis); or large particles such as
internalized receptors, viruses, bacteria, or bacterial toxins
(phagocytosis). In exocytosis, molecules are transported to the
cell surface. Exocytosis facilitates the placement or localization
of membrane-bound receptors or other membrane proteins and the
secretion of hormones, neurotransmitters, digestive enzymes, and
wastes. Endocytosis and exocytosis are fundamental to the function
of all types of cells.
[0042] Isolation of intracellular organelles from rat liver has
demonstrated the presence of two distinct organelles, the lysosome
and the peroxisome (de Duve, C. (1996) Ann. N.Y. Acad. Sci.
804:1-10). Lysosomes are the site of degradation of obsolete
intracellular material during autophagy and of extracellular
molecules following endocytosis and phagocytosis. They are derived
from endosomes, which in turn are formed from budding of the
trans-Golgi network or from clathrin-coated membrane vesicles
invaginating from the plasma membrane.
[0043] Protein sorting by transport vesicles, such as the endosome,
has important consequences for a variety of physiological processes
including cell surface growth, the biogenesis of distinct
intracellular organelles, endocytosis, and the controlled release
of hormones and neurotransmitters (Rothman, J. E. and Wieland, F.
T. (1996) Science 272:227-234). In particular, neurodegenerative
disorders and other neuronal pathologies are associated with
biochemical flaws during endosomal protein sorting or endosomal
biogenesis (Mayer R. J. et al. (1996) Adv. Exp. Med. Biol.
389:261-269).
[0044] Peroxisomes are independent organelles and are not members
of the secretory pathway family of organelles. They are
characterized by a single membrane and a finely granulated matrix
and are the site of many peroxide-generating oxidative reactions in
the cell. Peroxisomes are unique among eukaryotic organelles in
that their size, number, and enzyme content vary depending upon
organism, cell type, and metabolic needs. The majority of
peroxisome-associated proteins are membrane-bound or are found
proximal to the cytosolic or the lumenal side of the peroxisome
membrane (Waterham, H. R. and Cregg, J. M. (1996) BioEssays
19:57-66).
[0045] Genetic defects in peroxisome proteins which result in
peroxisomal deficiencies have been linked to a number of human
pathologies, including Zellweger syndrome, rhizomelic
chonrodysplasia punctata, X-linked adrenoleukodystrophy, acyl-CoA
oxidase deficiency, bifunctional enzyme deficiency, classical
Refsum's disease, DHAP alkyl transferase deficiency, and
acatalasemia (Moser, H. W. and Moser, A. B. (1996) Ann. NY Acad.
Sci. 804:427-441). In addition, Gartner, J. et al. (1991; Pediatr.
Res. 29:141-146) found a 22 kDa integral membrane protein
associated with lower density peroxisome-like subcellular fractions
in patients with Zellweger syndrome.
[0046] Normal embryonic development and control of germ cell
maturation is modulated by a number of secretory proteins which
interact with their respective membrane-bound receptor. Cell fate
during embryonic development is determined, for example, by members
of the activin/TGF-.beta. superfamily, cadherin(s), IGF-2, and
other morphogen(s). In addition, proliferation, maturation, and
redifferentiation of germ cell and reproductive tissues are
regulated, for example, by IGF-2, inhibin(s), activin(s), and
follistatin(s) (Petraglia, F. (1997) Placenta 18:3-8; and Mather,
J. P. et al. (1997) Proc. Soc. Exp. Biol. Med. 215:209-222).
[0047] Lymphocyte and Leukocyte Membrane Proteins
[0048] The B-cell response to antigens, which is modulated through
receptors, is an essential component of the normal immune system.
Mature B cells recognize foreign antigens through B cell receptors
(BCR) and produce specific antibodies which bind the foreign
antigens. The antigen/receptor complex is internalized, and the
antigen is proteolytically processed. To generate an efficient
response to complex antigens, the assistance of the BCR, BCR
associated proteins, and T cell is required. A small part of the
antigen remains complexed with major histocompatability complex-II
(MHCII) molecules on the surface of the B cells where the complex
can be recognized by T cells. MHCI molecules are on the surface of
non-lymphoid tissue and present antigens to macrophages or other
lymphoid cell types. T cells recognize and are activated by the
MHCI-antigen complex through interactions with the T cell
receptor/CD3 complex, a T cell surface multimeric, multiphenotypic
protein located in the plasma membrane. T cells activated by
antigen presentation secrete a variety of lymphokines that induce B
cell maturation, activate macrophages, and kill target cells.
[0049] Leukocytes have a fundamental role in the inflammatory and
immune resonse, and include monocytes/macrophages, mast cells,
polymorphonucleoleukocytes, natural killer cells, nuetrophils,
eosinophils, basophils, and myeloid precursors. Leukocyte membrane
proteins include members of the CD antigens, N-CAM, I-CAM, human
leukocyte antigen (HLA) class I and HLA class II gene products,
immunoglobulins, immunoglobulin receptors, complement, complement
receptors, interferons, interferon receptors, interleukin
receptors, and chemokine receptors.
[0050] Abnormal lymphocyte and leukocyte activity has been
associated with, for example, AIDS, immune hypersensitivity,
leukemias, leukopenia, systemic lupus, granulomatous disease, and
eosinophilia.
[0051] Apoptosis
[0052] A variety of ligands and their cellular receptors, enzymes,
tumor suppressors, viral gene products, pharmacological agents, and
inorganic ions have important positive or negative roles in
regulating and implementing the apoptotic destruction of a cell.
Although some specific components of the apoptotic pathway have
been identified and characterized, many interactions between the
proteins involved are undefined, leaving major aspects of the
pathway unknown.
[0053] A requirement for calcium in apoptosis was previously
suggested by studies showing the involvement of calcium levels in
DNA cleavage and Fas-mediated cell death (Hewish, D. R. and L. A.
Burgoyne (1973) Biochem. Biophys. Res. Commun. 52:504-510; Vignaux,
F. et al. (1995) J. Exp. Med. 181:781-786; and Oshimi, Y. and S.
Miyazaki (1995) J. Immunol. 154:599-609). Other studies show that
intracellular calcium concentrations increase when apoptosis is
triggered in thymocytes by either T cell receptor cross-linking or
by glucocorticoids, and cell death can be prevented by blocking
this increase (McConkey, D. J. et al. (1989) J. Immunol.
143:1801-1806; and McConkey, D. J. et al. (1989) Arch. Biochem.
Biophys. 269:365-370).
[0054] The discovery of new human membrane spanning proteins and
the polynucleotides encoding it satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
treatment, and prevention of neoplastic, immunological, and
reproductive disorders.
SUMMARY OF THE INVENTION
[0055] The invention features a substantially purified polypeptide,
human membrane spanning proteins (MSPs), having an amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and
SEQ ID NO:17 (SEQ ID NO:1 through SEQ ID NO:17), or fragments
thereof.
[0056] The invention further provides a substantially purified
variant having at least 90% amino acid identity to the amino acid
sequences of SEQ ID NO:1 through SEQ ID NO:17, or to a fragment of
any of these sequences. The invention also provides an isolated and
purified polynucleotide encoding the polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:1 through SEQ ID NO:17, or fragments thereof. The invention also
includes an isolated and purified polynucleotide variant having at
least 90% polynucleotide seqeunce identity to the polynucleotide
encoding the polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:1 through SEQ ID NO:17, or
fragments thereof.
[0057] Additionally, the invention provides an isolated and
purified polynucleotide which hybridizes under stringent conditions
to the polynucleotide encoding the polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1
through SEQ ID NO:17, or fragments thereof, as well as an isolated
and purified polynucleotide having a sequence which is
complementary to the polynucleotide encoding the polypeptide
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO:1 through SEQ ID NO:17, or fragments
thereof.
[0058] The invention also provides an isolated and purified
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ
ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID
NO:34 (SEQ ID NO:18 through SEQ ID NO:34), or fragments thereof.
The invention further provides an isolated and purified
polynucleotide variant having at least 90% polynucleotide sequence
identity to the polynucleotide sequence comprising a polynucleotide
sequence selected from the group consisting of SEQ ID NO:18 through
SEQ ID NO:34, or fragment thereof, as well as an isolated and
purified polynucleotide having a sequence which is complementary to
the polynucleotide comprising a polynucleotide sequence selected
from the group consisting of SEQ ID NO:18 through SEQ ID NO:34, or
fragments thereof.
[0059] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1 through SEQ ID NO:17, or fragments
thereof. In another aspect, the expression vector is contained
within a host cell.
[0060] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:1 through SEQ ID NO:17, or fragments
thereof, the method comprising the steps of: (a) culturing the host
cell containing an expression vector containing at least a fragment
of a polynucleotide encoding the polypeptide under conditions
suitable for the expression of the polypeptide; and (b) recovering
the polypeptide from the host cell culture.
[0061] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the amino
acid sequence selected from the group consisting of SEQ ID NO:1
through SEQ ID NO:17, or fragments thereof in conjunction with a
suitable pharmaceutical carrier.
[0062] The invention further includes a purified antibody which
binds to a polypeptide comprising the amino acid sequence selected
from the group consisting of SEQ ID NO:1 through SEQ ID NO:17, or
fragments thereof, as well as a purified agonist and a purified
antagonist to the polypeptide.
[0063] The invention also provides a method for treating or
preventing a neoplastic disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1 through
SEQ ID NO:17, or fragments thereof.
[0064] The invention also provides a method for treating or
preventing an immunological disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1 through
SEQ ID NO:17, or fragments thereof.
[0065] The invention also provides a method for treating or
preventing a reproductive disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1 through
SEQ ID NO:17, or fragments thereof.
[0066] The invention also provides a method for detecting a
polynucleotide encoding the polypeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:1 through
SEQ ID NO:17, or fragments thereof in a biological sample
containing nucleic acids, the method comprising the steps of: (a)
hybridizing the complement of the polynucleotide sequence encoding
the polypeptide comprising the amino acid sequence selected from
the group consisting of SEQ ID NO:1 through SEQ ID NO:17, or
fragments thereof to at least one of the nucleic acids of the
biological sample, thereby forming a hybridization complex; and (b)
detecting the hybridization complex, wherein the presence of the
hybridization complex correlates with the presence of a
polynucleotide encoding the polypeptide in the biological sample.
In one aspect, the nucleic acids of the biological sample are
amplified by the polymerase chain reaction prior to the hybridizing
step.
DESCRIPTION OF THE INVENTION
[0067] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described, as these may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0068] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0069] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and which
might be used in connection with the invention. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
[0070] DEFINITIONS
[0071] "MSP," as used herein, refers to the amino acid sequences of
substantially purified MSP obtained from any species, particularly
a mammalian species, including bovine, ovine, porcine, murine,
equine, and preferably the human species, from any source, whether
natural, synthetic, semi-synthetic, or recombinant.
[0072] The term "agonist," as used herein, refers to a molecule
which, when bound to MSP, increases or prolongs the duration of the
effect of MSP. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of MSP.
[0073] An "allele" or an "allelic sequence," as these terms are
used herein, is an alternative form of the gene encoding MSP.
Alleles may result from at least one mutation in the nucleic acid
sequence and may result in altered mRNAs or in polypeptides whose
structure or function may or may not be altered. Any given natural
or recombinant gene may have none, one, or many allelic forms.
Common mutational changes which give rise to alleles are generally
ascribed to natural deletions, additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in
combination with the others, one or more times in a given
sequence.
[0074] "Altered" nucleic acid sequences encoding MSP, as described
herein, include those sequences with deletions, insertions, or
substitutions of different nucleotides, resulting in a
polynucleotide the same MSP or a polypeptide with at least one
functional characteristic of MSP. Included within this definition
are polymorphisms which may or may not be readily detectable using
a particular oligonucleotide probe of the polynucleotide encoding
MSP, and improper or unexpected hybridization to alleles, with a
locus other than the normal chromosomal locus for the
polynucleotide sequence encoding MSP. The encoded protein may also
be "altered," and may contain deletions, insertions, or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent MSP. Deliberate amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues, as long as the biological or
immunological activity of MSP is retained. For example, negatively
charged amino acids may include aspartic acid and glutamic acid,
positively charged amino acids may include lysine and arginine, and
amino acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; and phenylalanine and tyrosine.
[0075] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In this context, "fragments", "immunogenic
fragments", or "antigenic fragments" refer to fragments of MSP
which are preferably about 5 to about 15 amino acids in length and
which retain some biological activity or immunological activity of
MSP. Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms are not meant to limit the
amino acid sequence to the complete native amino acid sequence
associated with the recited protein molecule.
[0076] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art (See, e.g., Dieffenbach, C. W.
and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold
Spring Harbor Press, Plainview N.Y., pp.1-5.)
[0077] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to MSP, decreases the amount or the
duration of the effect of the biological or immunological activity
of MSP. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of MSP.
[0078] As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fa,
F(ab').sub.2, and Fv fragments, which are capable of binding the
epitopic determinant. Antibodies that bind MSP polypeptides can be
prepared using intact polypeptides or using fragments containing
small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used to immunize an animal (e.g., a
mouse, a rat, or a rabbit) can be derived from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier
protein if desired. Commonly used carriers that are chemically
coupled to peptides include bovine serum albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then
used to immunize the animal.
[0079] The term "antigenic determinant," as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or a fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0080] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to a specific nucleic acid sequence. The term
"antisense strand" is used in reference to a nucleic acid strand
that is complementary to the "sense" strand. Antisense molecules
may be produced by any method including synthesis or transcription.
Once introduced into a cell, the complementary nucleotides combine
with natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0081] As used herein, the term "biologically active," refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
MSP, or of any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0082] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A." Complementarity between two single-stranded molecules may
be "partial," such that only some of the nucleic acids bind, or it
may be "complete," such that total complementarity exists between
the single stranded molecules. The degree of complementarity
between nucleic acid strands has significant effects on the
efficiency and strength of the hybridization between the nucleic
acid strands. This is of particular importance in amplification
reactions, which depend upon binding between nucleic acids strands,
and in the design and use of peptide nucleic acid (PNA)
molecules.
[0083] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence," as these
terms are used herein, refer broadly to any composition containing
the given polynucleotide or amino acid sequence. The composition
may comprise a dry formulation, an aqueous solution, or a sterile
composition. Compositions comprising polynucleotide sequences
encoding MSP or fragments of MSP may be employed as hybridization
probes. The probes may be stored in freeze-dried form and may be
associated with a stabilizing agent such as a carbohydrate. In
hybridizations, the probe may be deployed in an aqueous solution
containing salts (e.g., NaCl), detergents (e.g., SDS), and other
components (e.g., Denhardt's solution, dry milk, salmon sperm DNA,
etc.).
[0084] The phrase "consensus sequence," as used herein, refers to a
nucleic acid sequence which has been resequenced to resolve
uncalled bases, extended using XL-PCR (Perkin Elmer, Norwalk,
Conn.) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from the overlapping sequences of more than one
Incyte Clone using a computer program for fragment assembly, such
as the GELVIEW Fragment Assembly system (GCG, Madison, Wis.). Some
sequences have been both extended and assembled to produce the
consensus sequence.
[0085] As used herein, the term "correlates with expression of a
polynucleotide" indicates that the detection of the presence of
nucleic acids, the same or related to a nucleic acid sequence
encoding MSP, by northern analysis is indicative of the presence of
nucleic acids encoding MSP in a sample, and thereby correlates with
expression of the transcript from the polynucleotide encoding
MSP.
[0086] A "deletion," as the term is used herein, refers to a change
in the amino acid or nucleotide sequence that results in the
absence of one or more amino acid residues or nucleotides.
[0087] The term "derivative," as used herein, refers to the
chemical modification of MSP, of a polynucleotide sequence encoding
MSP, or of a polynucleotide sequence complementary to a
polynucleotide sequence encoding MSP. Chemical modifications of a
polynucleotide sequence can include, for example, replacement of
hydrogen by an alkyl, acyl, or amino group. A derivative
polynucleotide encodes a polypeptide which retains at least one
biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation,
pegylation, or any similar process that retains at least one
biological or immunological function of the polypeptide from which
it was derived.
[0088] The term "homology," as used herein, refers to a degree of
complementarity. There may be partial homology or complete
homology. The word "identity" may substitute for the word
"homology." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially homologous."
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or northern blot, solution
hybridization, and the like) under conditions of reduced
stringency. A substantially homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous sequence to the target sequence under conditions of
reduced stringency. This is not to say that conditions of reduced
stringency are such that non-specific binding is permitted, as
reduced stringency conditions require that the binding of two
sequences to one another be a specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% homology or
identity). In the absence of non-specific binding, the
substantially homologous sequence or probe will not hybridize to
the second non-complementary target sequence.
[0089] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MegAlign program
(DNASTAR, Inc., Madison Wis.). The MegAlign program can create
alignments between two or more sequences according to different
methods, e.g., the Clustal method. (See, e.g., Higgins, D. G. and
P. M. Sharp (1988) Gene 73:237-244.) The Clustal algorithm groups
sequences into clusters by examining the distances between all
pairs. The clusters are aligned pairwise and then in groups. The
percentage similarity between two amino acid sequences, e.g.,
sequence A and sequence B, is calculated by dividing the length of
sequence A, minus the number of gap residues in sequence A, minus
the number of gap residues in sequence B, into the sum of the
residue matches between sequence A and sequence B, times one
hundred. Gaps of low or of no homology between the two amino acid
sequences are not included in determining percentage similarity.
Percent identity between nucleic acid sequences can also be counted
or calculated by other methods known in the art, e.g., the Jotun
Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol.
183:626-645.) Identity between sequences can also be determined by
other methods known in the art, e.g., by varying hybridization
conditions.
[0090] "Human artificial chromosomes" (HACs), as described herein,
are linear microchromosomes which may contain DNA sequences of
about 6 kb to 10 Mb in size, and which contain all of the elements
required for stable mitotic chromosome segregation and maintenance.
(See, e.g., Harrington, J. J. et al. (1997) Nat. Genet.
15:345-355.)
[0091] The term "humanized antibody," as used herein, refers to
antibody molecules in which the amino acid sequence in the
non-antigen binding regions has been altered so that the antibody
more closely resembles a human antibody, and still retains its
original binding ability.
[0092] "Hybridization," as the term is used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0093] As used herein, the term "hybridization complex" as used
herein, refers to a complex formed between two nucleic acid
sequences by virtue of the formation of hydrogen bonds between
complementary bases. A hybridization complex may be formed in
solution (e.g., C.sub.0t or R.sub.0t analysis) or formed between
one nucleic acid sequence present in solution and another nucleic
acid sequence immobilized on a solid support (e.g., paper,
membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells or their nucleic acids have
been fixed).
[0094] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0095] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0096] The term "microarray," as used herein, refers to an
arrangement of distinct polynucleotides arrayed on a substrate,
e.g., paper, nylon or any other type of membrane, filter, chip,
glass slide, or any other suitable solid support.
[0097] The terms "element" or "array element" as used herein in a
microarray context, refer to hybridizable polynucleotides arranged
on the surface of a substrate.
[0098] The term "modulate," as it appears herein, refers to a
change in the activity of MSP. For example, modulation may cause an
increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of MSP.
[0099] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to an oligonucleotide, nucleotide,
polynucleotide, or any fragment thereof, to DNA or RNA of genomic
or synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which are greater than about 60 nucleotides in length, and most
preferably are at least about 100 nucleotides, at least about 1000
nucleotides, or at least about 10,000 nucleotides in length.
[0100] The terms "operably associated" or "operably linked," as
used herein, refer to functionally related nucleic acid sequences.
A promoter is operably associated or operably linked with a coding
sequence if the promoter controls the transcription of the encoded
polypeptide. While operably associated or operably linked nucleic
acid sequences can be contiguous and in reading frame, certain
genetic elements, e.g., repressor genes, are not contiguously
linked to the encoded polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0101] The term "oligonucleotide," as used herein, refers to a
nucleic acid sequence of at least about 6 nucleotides to 60
nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20 to 25 nucleotides, which can be used in PCR
amplification or in a hybridization assay or microarray. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimer," "primer," "oligomer," and "probe," as these
terms are commonly defined in the art.
[0102] "Peptide nucleic acid" (PNA), as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least about 5 nucleotides in length linked to
a peptide backbone of amino acid residues ending in lysine. The
terminal lysine confers solubility to the composition. PNAs
preferentially bind complementary single stranded DNA and RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (See, e.g., Nielsen, P. E. et al. (1993)
Anticancer Drug Des. 8:53-63.)
[0103] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding MSP, or fragments thereof, or MSP itself, may comprise a
bodily fluid; an extract from a cell, chromosome, organelle, or
membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA,
in solution or bound to a solid support; a tissue; a tissue print;
etc.
[0104] As used herein, the terms "specific binding" or
"specifically binding" refer to that interaction between a protein
or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon the presence of a particular
structure of the protein, e.g., the antigenic determinant or
epitope, recognized by the binding molecule. For example, if an
antibody is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0105] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotide
sequences and the claimed polynucleotide sequences. Suitably
stringent conditions can be defined by, for example, the
concentrations of salt or formamide in the prehybridization and
hybridization solutions, or by the hybridization temperature, and
are well known in the art. In particular, stringency can be
increased by reducing the concentration of salt, increasing the
concentration of formamide, or raising the hybridization
temperature.
[0106] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide, 5X SSPE,
0.3% SDS, and 200 .mu.g/ml sheared and denatured salmon sperm DNA.
Hybridization could occur under reduced stringency conditions as
described above, but in 35% formamide at a reduced temperature of
35.degree. C. The temperature range corresponding to a particular
level of stringency can be further narrowed by calculating the
purine to pyrimidine ratio of the nucleic acid of interest and
adjusting the temperature accordingly. Variations on the above
ranges and conditions are well known in the art.
[0107] The term "substantially purified," as used herein, refers to
nucleic acid or amino acid sequences that are removed from their
natural environment and are isolated or separated, and are at least
about 60% free, preferably about 75% free, and most preferably
about 90% free from other components with which they are naturally
associated.
[0108] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0109] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell.
Transformation may occur under natural or artificial conditions
according to various methods well known in the art, and may rely on
any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is selected based on the type of host cell being
transformed and may include, but is not limited to, viral
infection, electroporation, heat shock, lipofection, and particle
bombardment. The term "transformed" cells includes stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome, as well as transiently transformed
cells which express the inserted DNA or RNA for limited periods of
time.
[0110] A "variant" of MSP, as used herein, refers to an amino acid
sequence that is altered by one or more amino acids. The variant
may have "conservative" changes, wherein a substituted amino acid
has similar structural or chemical properties (e.g., replacement of
leucine with isoleucine). More rarely, a variant may have
"nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
DNASTAR software.
[0111] THE INVENTION
[0112] The invention is based on the discovery of new human
membrane spanning proteins, collectively referred to as MSP and
individually as MSP-1, MSP-2, MSP-3, MSP-4, MSP-5, MSP-6, MSP-7,
MSP-8, MSP-9, MSP-10, MSP-11, MSP-12, MSP-13, MSP-14, MSP-15,
MSP-16, and MSP-17; the polynucleotides encoding MSP (SEQ ID NO:18
through SEQ ID NO:34); and the use of these compositions for the
diagnosis, treatment, or prevention of neoplastic, immunological,
and reproductive disorders. Table 1 shows the sequence
identification numbers, Incyte Clone identification number, cDNA
library, public database (PD) homolog sequence identifier and
homolog species description for each of the human membrane spanning
proteins disclosed herein.
[0113] Nucleic acids encoding the MSP-1 of the present invention
were first identified in Incyte Clone 77138 from the synovial
membrane tissue cDNA library (SYNORAB01) using a computer search
for amino acid sequence alignments. A consensus sequence, SEQ ID
NO:18, was derived from Incyte Clones 77138 (SYNORAB01), 3576995
(BRONNOT01), 1995355 (BRSTTUT03), and 1260677 (SYNORAT05).
[0114] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1. MSP-1 is 238
amino acids in length and has two potential N glycosylation sites
at residues N71 and N72; one potential cAMP- and cGMP-dependent
protein kinase phosphorylation site at residue S233; three
potential casein kinase II phosphorylation sites at residues T111,
T131, and T195; four potential protein kinase C phosphorylation
sites at residues T10, S44, T186, and S233; and the PMP-22/EMP/MP20
membrane protein family signature from about residue V205 to about
residue M232; a proline-rich region from about P26 to about P41;
and the G-protein alpha subunit signature from about R61 to about
T76. MSP-1 shares 63% identity with the dog mucin-type gp40 protein
(g1628360). In addition, the PMP-22/EMP/MP20 membrane protein
family signature is conserved between these molecules. A fragment
of SEQ ID NO:18 from about nucleotide 379 to about nucleotide 405
is useful for designing oligonucleotides or for use as a
hybridization probe. Northern analysis shows the expression of this
sequence in reproductive, nervous, and connective tissue cDNA
libraries. Approximately 56% of these libraries are associated with
neoplastic disorders and 23% with immune response.
[0115] Nucleic acids encoding the MSP-2 of the present invention
were first identified in Incyte Clone 1381884 from the brain cDNA
library (BRAITUT08) using a computer search for amino acid sequence
alignments. A consensus sequence, SEQ ID NO:19, was derived
1TABLE 1 Clone Protein Nucleotide ID Library PD Homolog Homolog
species SEQ ID NO:1 SEQ ID NO:18 77138 SYNORAIB01 g1628360 Canis
familiaris SEQ ID NO:2 SEQ ID NO:19 1381884 BRAITUT08 g927071 Homo
sapiens SEQ ID NO:3 SEQ ID NO:20 1427590 SINTBST01 g2059326 Mus
musculus SEQ ID NO:4 SEQ ID NO:21 1457779 COLNFET02 g765256 Homo
sapiens SEQ ID NO:5 SEQ ID NO:22 1481261 CORPNOT02 g177900 Homo
sapiens SEQ ID NO:6 SEQ ID NO:23 1487802 UCMCL5T01 g2529739 Homo
sapiens SEQ ID NO:7 SEQ ID NO:24 1718830 BLADNOT06 g415907
Saccharomyces cerevisiae SEQ ID NO:8 SEQ ID NO:25 1737775 COLNNOT22
g1184066 Bos taurus SEQ ID NO:9 SEQ ID NO:26 1794154 PROSTUT05
WO9640907-A1 Homo sapiens SEQ ID NO:10 SEQ ID NO:27 2027624
KERANOT02 g458939 Saccharomyces cerevisiae SEQ ID NO:11 SEQ ID
NO:28 2057213 BEPINOT01 g50599 Mus musculus SEQ ID NO:12 SEQ ID
NO:29 2073804 ISLTNOT01 g602370 Saccharomyces cerevisiae SEQ ID
NO:13 SEQ ID NO:30 2175401 ENDCNOT03 g624778 Mus musculus SEQ ID
NO:14 SEQ ID NO:31 2741580 BRSTTUT14 g2586350 Homo sapiens SEQ ID
NO:15 SEQ ID NO:32 2779610 OVARTUT03 g475006 Homo sapiens SEQ ID
NO:16 SEQ ID NO:33 2879792 UTRSTUT05 g309074 Mus musculus SEQ ID
NO:17 SEQ ID NO:34 3231062 COTRNOT01 g529228 Felis catus
[0116] from Incyte Clones 1381884 (BRAITUT08), 1862104 (PROSNOT19),
79810 (SYNORAB01), 2372040 (ADRENOT07), 1811401 (PROSTUT 12),
1811401 (PROSTUT12), and 1927265 (BRSTNOT02).
[0117] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:2. MSP-2 is 83
amino acids in length and has one potential N glycosylation site at
residue N80; one potential casein kinase II phosphorylation site at
residue T71; and the inhibin beta A chain signature from about
residue E39 to about residue K60. MSP-2 shares 51% identity with
human thyroid receptor interactor (g927071). In addition, the
C-terminal glycine residue spacings
G41-{5N}-G47-{2N}-G50-{8N}-G59-{2N}-G62-{5N}-G69, where N is any
amino acid, are conserved between these molecules. A fragment of
SEQ ID NO:19 from about nucleotide 355 to about nucleotide 375 is
useful for designing oligonucleotides or for use as a hybridization
probe. Northern analysis shows the expression of this sequence in
reproductive, cardiovascular, nervous, and gastrointestinal cDNA
libraries. Approximately 43% of these libraries are associated with
neoplastic disorders and 21% with immune response.
[0118] Nucleic acids encoding the MSP-3 of the present invention
were first identified in Incyte Clone 1427590 from the ileum cDNA
library (SINTBST01) using a computer search for amino acid sequence
alignments. A consensus sequence, SEQ ID NO:20, was derived from
Incyte Clones 1427590 (SINTBST01), 2535753 (BRAINOT18), 1728970
(BRSTTUT08), 1596989 (BRAINOT14), 1505713 (BRAITUT07), 918144 and
1967560 (BRSTNOT04), 2817264 (BRSTNOT14), 732336 (LUNGNOT03), and
519913 (MMLR2DT01).
[0119] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:3. MSP-3 is 495
amino acids in length and has six potential N glycosylation sites
at residues N28, N119, N161, N241, N295, and N413; seven potential
casein kinase II phosphorylation sites at residues S110, T137,
T216, S338, S368, S456, and T462; six potential protein kinase C
phosphorylation sites at residues S322, S344, S392, S456, S467, and
S499; and the dopamine 1B receptor signature from about residue Q54
to about residue W67. MSP-3 shares 98% identity with the mouse
thymic epithelial cell surface antigen (g2059326). The cysteines at
C145, C308, C496, C507, C510, and C515 are conserved across both
molecules. A fragment of SEQ ID NO:20 from about nucleotide 1372 to
about nucleotide 1392 is useful for designing oligonucleotides or
for use as a hybridization probe. Northern analysis shows the
expression of this sequence in reproductive, nervous,
gastrointestinal, hematopoietic/immune, and cardiovascular cDNA
libraries. Approximately 49% of these libraries are associated with
neoplastic disorders and 21% with immune response.
[0120] Nucleic acids encoding the MSP-4 of the present invention
were first identified in Incyte Clone 1457779 from the fetal colon
cDNA library (COLNFET02) using a computer search for amino acid
sequence alignments. A consensus sequence, SEQ ID NO:21, was
derived from Incyte Clones 1457779 and 1457572 (COLNFET02), 2256441
(OVARTUT01), and the shotgun sequences SAGA01244, SAGA00103,
SAGA01339, SAGA00583, and SAGA01282.
[0121] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:4. MSP-4 is 495
amino acids in length and has seven potential N glycosylation sites
at residues N67, N135, N304, N325, N363, N374, and N447; one
potential cAMP- and cGMP-dependent protein kinase phosphorylation
site at residue S136; four potential casein kinase II
phosphorylation sites at residues S4, S153, T226, and T376; three
potential protein kinase C phosphorylation sites at residues S4,
S132, and T365; one potential tyrosine kinase phosphorylation site
at residue Y392; a potential signal peptide sequence from about
residue M1 to about G21; ten cysteine residues from about C78 to
about C403; and four GDA1/CD39 family transmembrane signatures: TM1
extends from about F43 to about Y59; TM2, from about T118 to about
L131; TM3, from about L162 to about L183; and TM4, from about G202
to about F215. MSP-4 shares 46% identity with human CD39 homolog
(g765256). The cysteines at C78, C102, C246, C273, C292, C316,
C329, C335, C381, and C403 are conserved across both molecules. In
addition, the hydrophobic transmembrane domains are conserved
between these molecules. A fragment of SEQ ID NO:21 from about
nucleotide 1543 to about nucleotide 1566 is useful for designing
oligonucleotides or for use as a hybridization probe. Northern
analysis shows the expression of this sequence in gastrointestinal
and ovarian cDNA libraries. Approximately 71% of these libraries
are associated with neoplastic disorders and 14% with immune
response.
[0122] Nucleic acids encoding the MSP-5 of the present invention
were first identified in Incyte Clone 1481261 from the corpus
callosum cDNA library (CORPNOT02) using a computer search for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:22, was
derived from Incyte Clones 1481261 and 1476703 (CORPNOT02), and
2963217 (SCORNOT04).
[0123] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:5. MSP-5 is 156
amino acids in length and has three potential casein kinase II
phosphorylation sites at residues S3, T114, and S151; three
potential protein kinase C phosphorylation sites at residues S3,
S32, and T114; and a potential GPR1/FUN34/yaaH family transmembrane
domain from about residue F95 to about residue I141. MSP-5 shares
25% identity with human differentiation-dependent intestinal A4
transmembrane protein (g177900). In addition, the hydrophobic
transmembrane domain and one potential protein kinase C
phosphorylation site are conserved between these molecules. A
fragment of SEQ ID NO:22 from about nucleotide 373 to about
nucleotide 396 is useful for designing oligonucleotides or for use
as a hybridization probe. Northern analysis shows the expression of
this sequence in brain, spinal cord, reproductive, muscle, and
Alzheimer's disease brain tissue cDNA libraries. Approximately 64%
of these libraries are associated with neoplastic disorders and 19%
with immune response.
[0124] Nucleic acids encoding the MSP-6 of the present invention
were first identified in Incyte Clone 1487802 from the umbilical
cord mononuclear cell cDNA library (UCMCL5T01) using a computer
search for amino acid sequence alignments. A consensus sequence,
SEQ ID NO:23, was derived from Incyte Clones 1487802 (UCMCL5T01),
1802847 (COLNNOT27), 613194 (COLNTUT02), 875949 (LUNGAST01), 506242
(TMLR3DT01), and 1939036 (HIPONOT01).
[0125] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:6. MSP-6 is 358
amino acids in length and has one potential N glycosylation site at
residue N324; one potential cAMP- and cGMP-dependent protein kinase
phosphorylation site at residue S340; six potential casein kinase
II phosphorylation sites at residues S46, S143, S186, T260, T293,
and S315; one potential glycosaminoglycan attachment site at
residue S162; and six potential protein kinase C phosphorylation
sites at residues T156, S170, S280, S339, S340, and S349. MSP-6
shares 95% identity with the human EBP50 protein (g2529739). A
fragment of SEQ ID NO:23 from about nucleotide 487 to about
nucleotide 516 is useful for designing oligonucleotides or for use
as a hybridization probe. Northern analysis shows the expression of
this sequence in reproductive, gastrointestinal, nervous, and
hematopoietic/immunologic cDNA libraries. Approximately 51% of
these libraries are associated with neoplastic disorders and 26%
with immune response.
[0126] Nucleic acids encoding the MSP-7 of the present invention
were first identified in Incyte Clone 1718830 from the bladder cDNA
library (BLADNOT06) using a computer search for amino acid sequence
alignments. A consensus sequence, SEQ ID NO:24, was derived from
Incyte Clones 1718830 (BLADNOT06), 2057919 (BEPINOT01), 3419169
(UCMCNOT04), 3236861 (COLNUCT03), and 2585037 (BRAITUT22).
[0127] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:7. MSP-7 is 280
amino acids in length and has one potential N glycosylation site at
residue N250; four potential casein kinase II phosphorylation sites
at residues T10, S70, S225, and S244; five potential protein kinase
C phosphorylation sites at residues T10, S144, S236, S244, and
T252; and the neutral zinc metallopeptidase zinc-binding region
signature from about residue L80 to about residue V89. MSP-7 shares
21% identity with the Saccharomyces cerevisiae protein encoded by
the YKR407 gene (g415907). In addition, the neutral zinc-binding
region signature is conserved between these molecules. The fragment
of SEQ ID NO:24 from about nucleotide 1006 to about nucleotide 1035
is useful for designing oligonucleotides or for use as a
hybridization probe. Northern analysis shows the expression of this
sequence in reproductive, urologic, cardiovascular, and
hematopoietic/immunologic cDNA libraries. Approximately 41% of
these libraries are associated with neoplastic disorders and 26%
with immune response.
[0128] Nucleic acids encoding the MSP-8 of the present invention
were first identified in Incyte Clone 1737775 from the colon cDNA
library (COLNNOT22) using a computer search for amino acid sequence
alignments. A consensus sequence, SEQ ID NO:25, was derived from
Incyte Clones 1737775 and 1734248 (COLNNOT22), 1578253 (DUODNOT01),
2214423 (SINTFET03), 608819 (COLNNOT01), 1629002 and 1626949
(COLNPOT01), 1498226 and 1429362 (SINTBST01), 2925341 (SININOT04),
and the shotgun sequences SAEC10115, SAEA00623, and SAEA00834.
[0129] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:8. MSP-8 is 914
amino acids in length and has eight potential N glycosylation sites
at residues N503, N585, N770, N804, N810, N831, N836, and N890; two
potential cAMP- and cGMP-dependent protein kinase phosphorylation
sites at residues T211 and T286; twelve potential casein kinase II
phosphorylation sites at residues T87, S245, S271, S364, S366,
T411, T597, T652, T663, S795, T870, and S876; one potential
glycosaminoglycan attachment site at residue S477; thirteen
potential protein kinase C phosphorylation sites at residues T68,
T84, T98, T207, T232, S366, S483, T563, T580, T594, T597, T601, and
S672; eleven cysteine residues from about C125 to about C421; a
potential signal peptide sequence from about residue M1 to about
S21; and a cadherin family signature from about N29 to about P44.
MSP-8 shares 53% identity with the bovine calcium-activated
chloride channel (g 1184066). The cysteines at C125, C187, C200,
C205, C210, C223, C250, C267, C308, C386, and C421 are conserved
across both molecules. A fragment of SEQ ID NO:25 from about
nucleotide 1447 to about nucleotide 1485 is useful for designing
oligonucleotides or for use as a hybridization probe. Northern
analysis shows the expression of this sequence in gastrointestinal,
female reproductive, and cardiovascular cDNA libraries.
Approximately 32% of these libraries are associated with neoplastic
disorders and 56% with immune response.
[0130] Nucleic acids encoding the MSP-9 of the present invention
were first identified in Incyte Clone 1794154 from the prostate
cDNA library (PROSTUT05) using a computer search for amino acid
sequence alignments. A consensus sequence, SEQ ID NO:26, was
derived from Incyte Clones 1794154 (PROSTUT05), 3116657
(LUNGTUT13), 1726333 (PROSNOT14), 3217158 (TESTNOT07), 1435295
(PANCNOT08), 2530829 (GBLANOT02), 728143 (SYNOOAT01), 1539910
(SINTTUT01), 1467860 (PANCTUT02), 1484333 (CORPNOT02), 1927469
(BRSTNOT02), 1516116 (PANCTUT01), 1571055 (UTRSNOT05), 1965064
(BRSTNOT04), and 2683922 (LUNGNOT23).
[0131] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:9. MSP-9 is 950
amino acids in length and has three potential N glycosylation site
at residues N667 N668 N835; six potential cAMP- and cGMP-dependent
protein kinase phosphorylation sites at residues S79, T204, S409,
S434, S658, and S767; seventeen potential casein kinase II
phosphorylation sites at residues S64, T65, S113, S284, T398, S409,
S418, T437, T449, S518, S576, T591, S611, T708, T717, S735, and
S838; twenty five potential protein kinase C phosphorylation sites
at residues T2, T28, S45, T55, S113, S124, S308, T347, T365, T384,
S393, T404, S418, T429, T430, S454, S537, S565, S572, S583, T600,
S607, T654, T670, and T732; one potential tyrosine kinase
phosphorylation site at residue Y918; and a potential signal
peptide sequence from about M1 to P23. MSP-9 shares 28% identity
with human type I, p80 IL-1-receptor intracellular domain ligand
(WO9640907-A1). A fragment of SEQ ID NO:26 from about nucleotide
3220 to about nucleotide 3243 is useful for designing
oligonucleotides or for use as a hybridization probe. Northern
analysis shows the expression of this sequence in gastrointestinal,
male reproductive, and muscle cDNA libraries. Approximately 54% of
these libraries are associated with neoplastic disorders and 22%
with immune response.
[0132] Nucleic acids encoding the MSP-10 of the present invention
were first identified in Incyte Clone 2027624 from the breast
epidermal keratinocyte cDNA library (KERANOT02) using a computer
search for amino acid sequence alignments. A consensus sequence,
SEQ ID NO:27, was derived from Incyte Clones 2027624 (KERANOT02),
3616164 (EPIPNOT01), 2492955 (ADRETUT05), 1557177 (BLADTUT04),
1466958 (PANCTUT02), 842091 (PROSTUT05), 1305186 (PLACNOT02),
1871863 (LEUKNOT02), 1345853 (PROSNOT11), and 1351147
(LATRTUT02).
[0133] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:10. MSP-10 is 578
amino acids in length and has three potential N glycosylation sites
at residues N164, N291, and N327; nine potential casein kinase II
phosphorylation sites at residues S29, T52, S81, S108, T119, T121,
S126, T267, and T303; one potential glycosaminoglycan attachment
site at residue S359; seven potential protein kinase C
phosphorylation sites at residues S29, S81, S126, S156, S196, T311,
and T403; one potential tyrosine kinase phosphorylation site at
residue Y287; and one potential signal peptide sequence from about
M1 to about A23. MSP-10 shares 24% identity with Saccharomyces
cerevisiae protein encoded by YHR188c gene (g458939). Fragments of
SEQ ID NO:27 from about nucleotide 1078 to about nucleotide 1101
and from about nucleotide 1585 to about nucleotide 1614 are useful
for designing oligonucleotides or for use as a hybridization probe.
Northern analysis shows the expression of this sequence in
reproductive, gastrointestinal, nervous, and cardiovascular cDNA
libraries. Approximately 50% of these libraries are associated with
neoplastic disorders and 24% with immune response.
[0134] Nucleic acids encoding the MSP-11 of the present invention
were first identified in Incyte Clone 2057213 from the bronchial
epithelium cell line cDNA library (BEPINOT01) using a computer
search for amino acid sequence alignments. A consensus sequence,
SEQ ID NO:28, was derived from Incyte Clones 2057213 (BEPINOT01),
2252506 (OVARTUT01), 3431316 (SKINNOT04), and 2670856
(ESOGTUT02).
[0135] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:11. MSP-11 is 270
amino acids in length and has one potential casein kinase II
phosphorylation site at residue T202; two potential protein kinase
C phosphorylation sites at residues T182 and T238; four potential
membrane spanning domains: TM1 extends from about Q48 to about I71;
TM2, from about I74 to about Y97; TM3, from about G125 to about
154; and TM4, from about A165 to about L209; and the connexin 1
signature from about C53 to about D66 and the connexin 2 signature
from about C164 to about P181. MSP-11 shares 81% identity with
mouse connexin 31 (g50599). In addition, the hydrophobic
transmembrane domains are conserved between these molecules. A
fragment of SEQ ID NO:28 from about nucleotide 559 to about
nucleotide 588 is useful for designing oligonucleotides or for use
as a hybridization probe. Northern analysis shows the expression of
this sequence in gastrointestinal, reproductive, and bronchial
epithelial cell line cDNA libraries. Approximately 40% of these
libraries are associated with neoplastic disorders.
[0136] Nucleic acids encoding the MSP-12 of the present invention
were first identified in Incyte Clone 2073804 from the pancreatic
islelet cDNA library (ISLTNOT01) using a computer search for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:29, was
derived from Incyte Clones 2073804 (ISLTNOT01) and 1982628
(LUNGTUT03).
[0137] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:12. MSP-12 is 154
amino acids in length and has two potential casein kinase II
phosphorylation sites at residues T60 and T82; two potential
glycosaminoglycan attachment sites at residues S12 and S14; four
potential protein kinase C phosphorylation sites at residues S5,
T60, T67, and T82; and four potential transmembrane regions: TM1
from about K18 to about F30; TM2 from about H54 to about L61; TM3
from about M73 to about V83; and TM4 from about K145 to about S154.
MSP-12 shares 32% identity with the Saccharomyces cerevisiae
potential transmembrane protein encoded by the Ye10003wp gene
(g602370). A fragment of SEQ ID NO:29 from about nucleotide 223 to
about nucleotide 252 is useful for designing oligonucleotides or
for use as a hybridization probe. Northern analysis shows the
expression of this sequence in cardiovascular, reproductive, and
nervous tissue cDNA libraries. Approximately 62% of these libraries
are associated with neoplastic disorders and 31% with immune
response.
[0138] Nucleic acids encoding the MSP-13 of the present invention
were first identified in Incyte Clone 2175401 from the dermal
microvascular endothelial cell cDNA library (ENDCNOT03) using a
computer search for amino acid sequence alignments. A consensus
sequence, SEQ ID NO:30, was derived from Incyte Clones 2175401
(ENDCNOT03), 428432 (BLADNOT01), 2175401 (ENDCNOT03), 79481
(SYNORAB01), and 415866 (BRSTNOT01).
[0139] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:13. MSP-13 is 263
amino acids in length and has one potential N glycosylation site at
residue N166; three potential casein kinase II phosphorylation
sites at residues T35, S132, and S134; and six potential protein
kinase C phosphorylation sites at residues T28, T35, S50, S100,
S185, and S224. MSP-13 shares 95% identity with the mouse E25
integral membrane protein (g624778). A fragment of SEQ ID NO:30
from about nucleotide 415 to about nucleotide 444 is useful for
designing oligonucleotides or for use as a hybridization probe.
Northern analysis shows the expression of this sequence in
reproductive, nervous, cardiovascular, muscle, and in Alzheimer's
disease brain cDNA libraries. Approximately 41% of these libraries
are associated with neoplastic disorders and 30% with immune
response.
[0140] Nucleic acids encoding the MSP-14 of the present invention
were first identified in Incyte Clone 2741580 from the breast cDNA
library (BRSTTUT14) using a computer search for amino acid sequence
alignments. A consensus sequence, SEQ ID NO:31, was derived from
Incyte Clones 2741580 (BRSTTUT14), 2672813, (KIDNNOT19), 779308
(MYOMNOT01), and 1996070 (BRSTTUT03).
[0141] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:14. MSP-14 is 240
amino acids in length and has one potential N glycosylation site at
residue N180; one potential casein kinase II phosphorylation site
at residue T164; one potential protein kinase C phosphorylation
site at residue T233; and the TM4SF protein family signature from
about residue G61 to about residue L83. In addition, there are four
potential membrane spanning domains: TM1 extends from about L10 to
about L34; TM2, from about S50 to about I76; TM3, from about K77 to
about F105; and TM4, from about V204 to about I230. MSP-14 shares
54% identity with human NAG-2 TM4SF protein (g2586350). In
addition, the transmembrane domains are conserved between these
molecules. A fragment of SEQ ID NO:31 from about nucleotide 316 to
about nucleotide 348 is useful for designing oligonucleotides or
for use as a hybridization probe. Northern analysis shows the
expression of this sequence in gastrointestinal, reproductive, and
urologic cDNA libraries. Approximately 58% of these libraries are
associated with neoplastic disorders and 33% with immune
response.
[0142] Nucleic acids encoding the MSP-15 of the present invention
were first identified in Incyte Clone 2779610 from the ovarian cDNA
library (OVARTUT03) using a computer search for amino acid sequence
alignments. A consensus sequence, SEQ ID NO:32, was derived from
Incyte Clones 2779610 (OVARTUT03), 260635 and 483040 (HNT2RAT01),
762225 (BRAITUT02), 2014257 (TESTNOT03), 833639 (PROSNOT07), and
414915 (BRSTNOT01).
[0143] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:15. MSP-15 is 245
amino acids in length and has one potential N glycosylation site at
residue N134; two potential casein kinase II phosphorylation sites
at residues S135 and T203; four potential protein kinase C
phosphorylation sites at residues S5, T82, S121, and S169; one
potential tyrosine kinase phosphorylation site at residue Y167; and
the TM4SF protein family signature from about residue G66 to about
residue I237. In addition, there are three potential membrane
spanning domians: TM1 extends from about T15 to about G42; TM2,
from about V63 to about F112; TM3, from about I204 to about T238.
MSP-15 shares 48% identity with human TALLA-1 TM4SF protein
(g475006). In addition, the transmembrane domains are conserved
between these molecules. A fragment of SEQ ID NO:32 from about
nucleotide 319 to about nucleotide 351 is useful for designing
oligonucleotides or for use as a hybridization probe. Northern
analysis shows the expression of this sequence in gastrointestinal,
reproductive, nervous, and urologic cDNA libraries. Approximately
55% of these libraries are associated with neoplastic disorders and
16% with immune response.
[0144] Nucleic acids encoding the MSP-16 of the present invention
were first identified in Incyte Clone 2879792 from the uterus cDNA
library (UTRSTUT05) using a computer search for amino acid sequence
alignments. A consensus sequence, SEQ ID NO:33, was derived from
Incyte Clones 2879792 (UTRSTUT05), 693310 (LUNGNOT23), 358525
(SYNORAB01), and 1822710 (GBLATUT01).
[0145] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:16. MSP-16 is 275
amino acids in length and has one potential N glycosylation site at
residue N140; one potential casein kinase II phosphorylation site
at residue S271; a potential signal peptide sequence from about
residue M1 to about residue S25; and potential transmembrane
regions from about residue L-79 to about residue C112 and from
about residue L148 to about residue A167. MSP-16 shares 64%
identity with the mouse 19.5 cell surface protein (g309074). In
addition, the hydrophobic transmembrane domains are conserved
between these molecules. A fragment of SEQ ID NO:33 from about
nucleotide 607 to about nucleotide 639 is useful for designing
oligonucleotides or for use as a hybridization probe. Northern
analysis shows the expression of this sequence in gastrointestinal,
reproductive, nervous, dermatological, and cardiovascular cDNA
libraries. Approximately 43% of these libraries are associated with
neoplastic disorders and 23% with immune response.
[0146] Nucleic acids encoding the MSP-17 of the present invention
were first identified in Incyte Clone 3231062 from the transverse
colon cDNA library (COTRNOT01) using a computer search for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:34, was
derived from Incyte Clones 3231062 (COTRNOT01), 3403088
(ESOGNOT03), 3523907 (ESOGTUN01), 3706862 and 3704223 (PENCNOT07),
3201831 (PENCNOT02), and 1555684 (BLADTUT04).
[0147] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:17. MSP-17 is 222
amino acids in length and has one potential N glycosylation site at
residue N139; two potential casein kinase II phosphorylation sites
at residues S46 and T122; one potential protein kinase C
phosphorylation site at residue S183; one potential tyrosine kinase
phosphorylation site at residue Y56; and the TM4SF protein family
signature from about residue G66 to about residue L88. In addition,
there are four potential transmembrane domains: TM1 extends from
about Y13 to about F36; TM2, from about F55 to about M76; TM3, from
about R82 to about F110; and TM4, from about L188 to about I203.
MSP-17 shares 46% identity with the feline CD9 TM4SF protein
(g529228). In addition, the hydrophobic transmembrane domains are
conserved between these molecules. A fragment of SEQ ID NO:34 from
about nucleotide 595 to about nucleotide 621 is useful for
designing oligonucleotides or for use as a hybridization probe.
Northern analysis shows the expression of this sequence in
gastrointestinal, male reproductive, and muscle cDNA libraries.
Approximately 55% of these libraries are associated with neoplastic
disorders and 18% with immune response.
[0148] The invention also encompasses MSP variants. A preferred MSP
variant is one which has at least about 80%, more preferably at
least about 90%, and most preferably at least about 95% amino acid
sequence identity to the MSP amino acid sequence, and which
contains at least one functional or structural characteristic of
MSP.
[0149] The invention also encompasses polynucleotides which encode
MSP. Accordingly, any nucleic acid sequence which encodes the amino
acid sequence of MSP can be used to produce recombinant molecules
which express MSP. In a particular embodiment, the invention
encompasses a polynucleotide consisting of a nucleic acid sequence
selected from the group consisting of SEQ ID NO:18 through SEQ ID
NO:34.
[0150] The invention also encompasses a variant of a polynucleotide
sequence encoding MSP. In particular, such a variant polynucleotide
sequence will have at least about 80%, more preferably at least
about 90%, and most preferably at least about 95% polynucleotide
sequence identity to the polynucleotide sequence encoding MSP. A
particular aspect of the invention encompasses a variant of a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:18 through SEQ ID NO:34 which has at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide consisting of a nucleic acid sequence selected
from the group consisting of SEQ ID NO:18 through SEQ ID NO:34. Any
one of the polynucleotide variants described above can encode an
amino acid sequence which contains at least one functional or
structural characteristic of MSP.
[0151] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding MSP, some bearing minimal
homology to the polynucleotide sequences of any known and naturally
occurring gene, may be produced. Thus, the invention contemplates
each and every possible variation of polynucleotide sequence that
could be made by selecting combinations based on possible codon
choices. These combinations are made in accordance with the
standard triplet genetic code as applied to the polynucleotide
sequence of naturally occurring MSP, and all such variations are to
be considered as being specifically disclosed.
[0152] Although nucleotide sequences which encode MSP and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring MSP under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding MSP or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding MSP and its derivatives without altering the
encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater
half-life, than transcripts produced from the naturally occurring
sequence.
[0153] The invention also encompasses production of DNA sequences
which encode MSP and MSP derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding MSP or any fragment thereof.
[0154] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequences, and in particular, those shown in SEQ ID NO:18 through
SEQ ID NO:34, and fragments thereof, under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399-407; and Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.)
[0155] Methods for DNA sequencing are well known and generally
available in the art and may be used to practice any of the
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, Sequenase.RTM. (US
Biochemical Corp., Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of polymerases and proofreading exonucleases such as
those found in the ELONGASE Amplification System (GIBCO/BRL,
Gaithersburg, Md.). Preferably, the process is automated with
machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,
Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,
Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin
Elmer).
[0156] The nucleic acid sequences encoding MSP may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences, such as
promoters and regulatory elements. For example, one method which
may be employed, restriction-site PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus. (See, e.g.,
Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) In particular,
genomic DNA is first amplified in the presence of a primer which is
complementary to a linker sequence within the vector and a primer
specific to a region of the nucleotide sequenc. The amplified
sequences are then subjected to a second round of PCR with the same
linker primer and another specific primer internal to the first
one. Products of each round of PCR are transcribed with an
appropriate RNA polymerase and sequenced using reverse
transcriptase.
[0157] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region. (See, e.g.,
Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) The primers
may be designed using commercially available software such as OLIGO
4.06 Primer Analysis software (National Biosciences Inc., Plymouth,
Minn.) or another appropriate program to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the target sequence at temperatures of about
68.degree. C. to 72.degree. C. The method uses several restriction
enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then circularized by intramolecular ligation
and used as a PCR template.
[0158] Another method which may be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In
this method, multiple restriction enzyme digestions and ligations
may be used to place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule before performing PCR. Other
methods which may be used to retrieve unknown sequences are known
in the art. (See, e.g., Parker, J. D. et al. (1991) Nucleic Acids
Res. 19:3055-3060.) Additionally, one may use PCR, nested primers,
and PROMOTERFINDER libraries to walk genomic DNA (Clontech, Palo
Alto, Calif.). This process avoids the need to screen libraries and
is useful in finding intron/exon junctions.
[0159] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into 5' non-transcribed regulatory regions.
[0160] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and a charge coupled device
camera for detection of the emitted wavelengths. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer),
and the entire process from loading of samples to computer analysis
and electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0161] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode MSP may be used in
recombinant DNA molecules to direct expression of MSP, or fragments
or functional equivalents thereof, in appropriate host cells. Due
to the inherent degeneracy of the genetic code, other DNA sequences
which encode substantially the same or a functionally equivalent
amino acid sequence may be produced, and these sequences may be
used to clone and express MSP.
[0162] As will be understood by those of skill in the art, it may
be advantageous to produce MSP-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0163] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter MSP-encoding sequences for a variety of reasons including,
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and
so forth.
[0164] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding MSP may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of MSP activity, it may
be useful to encode a chimeric MSP protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the MSP
encoding sequence and the heterologous protein sequence, so that
MSP may be cleaved and purified away from the heterologous
moiety.
[0165] In another embodiment, sequences encoding MSP may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucleic
Acids Symp. Ser. 7:215-223, and Horn, T. et al. (1980) Nucleic
Acids Symp. Ser. 7:225-232.) Alternatively, the protein itself may
be produced using chemical methods to synthesize the amino acid
sequence of MSP, or a fragment thereof. For example, peptide
synthesis can be performed using various solid-phase techniques.
(See, e.g., Roberge, J. Y. et al. (1995) Science 269:202-204.)
Automated synthesis may be achieved using the ABI 431A Peptide
Synthesizer (Perkin Elmer). Additionally, the amino acid sequence
of MSP, or any part thereof, may be altered during direct synthesis
and/or combined with sequences from other proteins, or any part
thereof, to produce a variant polypeptide.
[0166] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez, R. M. and
F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton, T. (1983) Proteins,
Structures and Molecular Properties, WH Freeman and Co., New York
N.Y.)
[0167] In order to express a biologically active MSP, the
nucleotide sequences encoding MSP or derivatives thereof may be
inserted into appropriate expression vector, i.e., a vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0168] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding MSP and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al. (1995, and
periodic supplements) Current Protocols in Molecular Biology, John
Wiley & Sons, New York N.Y., ch. 9, 13, and 16.)
[0169] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding MSP. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0170] The "control elements" or "regulatory sequences" are those
non-translated regions, e.g., enhancers, promoters, and 5' and 3'
untranslated regions, of the vector and polynucleotide sequences
encoding MSP which interact with host cellular proteins to carry
out transcription and translation. Such elements may vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used. For example, when cloning in bacterial systems, inducible
promoters, e.g., hybrid lacZ promoter of the BLUESCRIPT phagemid
(Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (GIBCO/BRL), may
be used. The baculovirus polyhedrin promoter may be used in insect
cells. Promoters or enhancers derived from the genomes of plant
cells (e.g., heat shock, RUBISCO, and storage protein genes) or
from plant viruses (e.g., viral promoters or leader sequences) may
be cloned into the vector. In mammalian cell systems, promoters
from mammalian genes or from mammalian viruses are preferable. If
it is necessary to generate a cell line that contains multiple
copies of the sequence encoding MSP, vectors based on SV40 or EBV
may be used with an appropriate selectable marker.
[0171] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for MSP. For example, when
large quantities of MSP are needed for the induction of antibodies,
vectors which direct high level expression of fusion proteins that
are readily purified may be used. Such vectors include, but are not
limited to, multifunctional E. coli cloning and expression vectors
such as BLUESCRIPT (Stratagene), in which the sequence encoding MSP
may be ligated into the vector in frame with sequences for the
amino-terminal Met and the subsequent 7 residues of
.beta.-galactosidase so that a hybrid protein is produced, and pIN
vectors. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J.
Biol. Chem. 264:5503-5509.) pGEX vectors (Pharmacia Biotech,
Uppsala, Sweden) may also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione.
Proteins made in such systems may be designed to include heparin,
thrombin, or factor XA protease cleavage sites so that the cloned
polypeptide of interest can be released from the GST moiety at
will.
[0172] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. (See, e.g., Ausubel,
supra; and Bitter, G. A. et al. (1987) Methods Enzymol.
153:516-544.)
[0173] In cases where plant expression vectors are used, the
expression of sequences encoding MSP may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.
6:307-311.) Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used. (See, e.g.,
Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results
Probl. Cell Differ. 17:85-105.) These constructs can be introduced
into plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews. (See, e.g., Hobbs, S. or Murry, L. E.
in McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill, New York N.Y.; pp. 191-196.)
[0174] An insect system may also be used to express MSP. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding MSP may be cloned into a non-essential region of
the virus, such as the polyhedrin gene, and placed under control of
the polyhedrin promoter. Successful insertion of sequences encoding
MSP will render the polyhedrin gene inactive and produce
recombinant virus lacking coat protein. The recombinant viruses may
then be used to infect, for example, S. frugiperda cells or
Trichoplusia larvae in which MSP may be expressed. (See, e.g.,
Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. USA
91:3224-3227.)
[0175] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding MSP may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing MSP in
infected host cells. (See, e.g., Logan, J. and T. Shenk (1984)
Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition,
transcription enhancers, such as the Rous sarcoma virus (RSV)
enhancer, may be used to increase expression in mammalian host
cells.
[0176] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of about 6 kb to 10 Mb are constructed and
delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0177] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding MSP. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding MSP and its initiation codon and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers appropriate for the particular cell
system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
[0178] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and WI38), are available from the American
Type Culture Collection (ATCC, Manassas Va.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0179] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing MSP can be transformed using
expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells may be allowed to grow for about 1 to 2 days in
enriched media before being switched to selective media. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.
[0180] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase genes and adenine
phosphoribosyltransferase genes, which can be employed in tk and
apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell
11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823) Also,
antimetabolite, antibiotic, or herbicide resistance can be used as
the basis for selection. For example, dhfr confers resistance to
methotrexate; npt confers resistance to the aminoglycosides
neomycin and G-418; and als or pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA
77:3567-3570; Colbere-Garapin, F. et al (1981) J. Mol. Biol.
150:1-14; and Murry, supra.) Additional selectable genes have been
described, e.g., trpB, which allows cells to utilize indole in
place of tryptophan, or hisD, which allows cells to utilize
histinol in place of histidine. (See, e.g., Hartman, S. C. and R.
C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.)
Visible markers, e.g., anthocyanins, .beta. glucuronidase and its
substrate GUS, luciferase and its substrate luciferin may be used.
Green fluorescent proteins (GFP) (Clontech, Palo Alto, Calif.) can
also be used. These markers can be used not only to identify
transformants, but also to quantify the amount of transient or
stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C. A. et al. (1995) Methods Mol. Biol.
55:121-131.)
[0181] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding MSP is inserted within a marker gene
sequence, transformed cells containing sequences encoding MSP can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding MSP under the control of a single promoter.
Expression of the marker gene in response to induction or selection
usually indicates expression of the tandem gene as well.
[0182] Alternatively, host cells which contain the nucleic acid
sequence encoding MSP and express MSP may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein
sequences.
[0183] The presence of polynucleotide sequences encoding MSP can be
detected by DNA-DNA or DNA-RNA hybridization or amplification using
probes or fragments or fragments of polynucleotides encoding MSP.
Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding MSP
to detect transformants containing DNA or RNA encoding MSP.
[0184] A variety of protocols for detecting and measuring the
expression of MSP, using either polyclonal or monoclonal antibodies
specific for the protein, are known in the art. Examples of such
techniques include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
MSP is preferred, but a competitive binding assay may be employed.
These and other assays are well described in the art. (See, e.g.,
Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual,
APS Press, St. Paul Minn., Section IV; and Maddox, D. E. et al.
(1983) J. Exp. Med. 158:1211-1216).
[0185] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding MSP include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding MSP, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
Suitable reporter molecules or labels which may be used for ease of
detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0186] Host cells transformed with nucleotide sequences encoding
MSP may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode MSP may be designed to
contain signal sequences which direct secretion of MSP through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding MSP to nucleotide sequences
encoding a polypeptide domain which will facilitate purification of
soluble proteins. Such purification facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences, such as those
specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.), between the purification domain and the MSP encoding
sequence may be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing MSP and a nucleic acid encoding 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification on immobilized metal ion
affinity chromatography (IMAC). (See, e.g., Porath, J. et al.
(1992) Prot. Exp. Purif. 3:263-281.) The enterokinase cleavage site
provides a means for purifying MSP from the fusion protein. (See,
e.g., Kroll, D. J. et al. (1993) DNA Cell Biol. 12:441-453.)
[0187] Fragments of MSP may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, T. E. (1984) Protein: Structures
and Molecular Properties, pp. 55-60, W.H. Freeman and Co., New York
N.Y.) Protein synthesis may be performed by manual techniques or by
automation. Automated synthesis may be achieved, for example, using
the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer).
Various fragments of MSP may be synthesized separately and then
combined to produce the full length molecule.
[0188] THERAPEUTICS
[0189] Chemical and structural homology exits among the human
membrane spanning proteins of the invention. In addition, the
expression of MSP is closely associated with cell proliferation,
and with tissues associated cancer, the immune response, and in
reproductive tissues. Therefore, MSP appears to play a role in
cancer, immunological, and reproductive disorders, in particular
where increased activity or synthesis appears to be associated with
these disorders.
[0190] In one embodiment, antagonists which decrease the expression
or activity of MSP may be administered to a subject to treat or
prevent a neoplastic disorder such as adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma. Such
cancers include, but are not limited to, cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver,
lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
In one aspect, antibodies which specifically bind MSP may be used
directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a pharmaceutical agent to cells or tissue
which express MSP.
[0191] In another embodiment, a vector expressing the complement of
the polynucleotide encoding MSP may be administered to a subject to
treat or prevent a neoplastic disorder including, but not limited
to, those listed above.
[0192] In yet another embodiment, antagonists which decrease the
activity of MSP may be administered to a subject to treat or
prevent an immunological disorder. Such immunological disorders may
be associated with conditions such as AIDS, Addison's disease,
adult respiratory distress syndrome, allergies, anemia, asthma,
atherosclerosis, bronchitis, cholecystitus, Crohn's disease,
ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout,
Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, rheumatoid arthritis, scleroderma,
Sjogren's syndrome, and autoimmune thyroiditis; complications of
cancer, hemodialysis, extracorporeal circulation; viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections; and
trauma. In one aspect, antibodies which specifically bind MSP may
be used directly as an antagonist or indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or
tissue which express MSP.
[0193] In another embodiment, a vector expressing the complement of
the polynucleotide encoding MSP may be administered to a subject to
treat or prevent an immunological disorder including, but not
limited to, those listed above.
[0194] In a further embodiment, an antagonist of MSP may be
administered to a subject to treat or prevent a reproductive
disorder. Such a reproductive disorder may include, but is not
limited to, disorders of prolactin production; infertility,
including tubal disease, ovulatory defects, and endometriosis;
disruptions of the estrous cycle, disruptions of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation
syndrome, endometrial and ovarian tumors, autoimmune disorders,
ectopic pregnancy, and teratogenesis; cancer of the breast, uterine
fibroids, fibrocystic breast disease, galactorrhea; disruptions of
spermatogenesis, abnormal sperm physiology, cancer of the testis,
cancer of the prostate, benign prostatic hyperplasia, prostatitis,
Peyronie's disease, carcinoma of the male breast and gynecomastia.
In one aspect, an antibody which specifically binds MSP may be used
directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a pharmaceutical agent to cells or tissue
which express MSP.
[0195] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding MSP may be administered
to a subject to treat or prevent a reproductive disorder including,
but not limited to, those described above.
[0196] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0197] An antagonist of MSP may be produced using methods which are
generally known in the art. In particular, purified MSP may be used
to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind MSP. Antibodies to
MSP may also be generated using methods that are well known in the
art. Such antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies (i.e., those which inhibit dimer formation)
are especially preferred for therapeutic use.
[0198] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with MSP or with any fragment or oligopeptide thereof
which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0199] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to MSP have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 10 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of MSP amino acids may be fused
with those of another protein, such as KLH, and antibodies to the
chimeric molecule may be produced.
[0200] Monoclonal antibodies to MSP may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. USA 80:2026-2030; and Cole, S. P. et al. (1984) Mol.
Cell Biol. 62:109-120.)
[0201] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. USA
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively,
techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce
MSP-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton, D. R. (1991) Proc.
Natl. Acad. Sci. USA 88:10134-10137.)
[0202] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; and Winter, G.
et al. (1991) Nature 349:293-299.)
[0203] Antibody fragments which contain specific binding sites for
MSP may also be generated. For example, such fragments include, but
are not limited to, F(ab')2 fragments produced by pepsin digestion
of the antibody molecule and Fab fragments generated by reducing
the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity. (See, e.g., Huse, W. D. et al. (1989) Science
246:1275-1281.)
[0204] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between MSP and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering MSP epitopes
is preferred, but a competitive binding assay may also be employed.
(Maddox, supra.)
[0205] In another embodiment of the invention, the polynucleotides
encoding MSP, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding MSP may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding MSP. Thus, complementary molecules or
fragments may be used to modulate MSP activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding MSP.
[0206] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors which
will express nucleic acid sequences complementary to the
polynucleotides of the gene encoding MSP. (See, e.g., Sambrook,
supra; and Ausubel, supra.)
[0207] Genes encoding MSP can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding MSP. Such constructs
may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in the absence of integration into the
DNA, such vectors may continue to transcribe RNA molecules until
they are disabled by endogenous nucleases. Transient expression may
last for a month or more with a non-replicating vector, and may
last even longer if appropriate replication elements are part of
the vector system.
[0208] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding MSP. Oligonucleotides derived from the
transcription initiation site, e.g., between about positions -10
and +10 from the start site, are preferred. Similarly, inhibition
can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful because it causes inhibition of the ability
of the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the
literature. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E.
and B. I. Carr, Molecular and Immunologic Approaches, Futura
Publishing Co., Mt. Kisco N.Y., pp. 163-177.) A complementary
sequence or antisense molecule may also be designed to block
translation of mRNA by preventing the transcript from binding to
ribosomes.
[0209] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding MSP.
[0210] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides, corresponding to the region of the target
gene containing the cleavage site, may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0211] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding MSP. Such DNA sequences may be incorporated into
a wide variety of vectors with suitable RNA polymerase promoters
such as T7 or SP6. Alternatively, these cDNA constructs that
synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines, cells, or tissues.
[0212] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule, or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0213] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nat. Biotechnol. 15:462-466.)
[0214] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0215] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of MSP, antibodies to MSP, and mimetics,
agonists, antagonists, or inhibitors of MSP. The compositions may
be administered alone or in combination with at least one other
agent, such as a stabilizing compound, which may be administered in
any sterile, biocompatible pharmaceutical carrier including, but
not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs, or hormones.
[0216] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0217] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton Pa.).
[0218] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0219] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0220] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0221] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0222] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0223] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0224] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0225] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0226] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of MSP, such labeling
would include amount, frequency, and method of administration.
[0227] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0228] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0229] A therapeutically effective dose refers to that amount of
active ingredient, for example MSP or fragments thereof, antibodies
of MSP, and agonists, antagonists or inhibitors of MSP, which
ameliorates the symptoms or condition. Therapeutic efficacy and
toxicity may be determined by standard pharmaceutical procedures in
cell cultures or with experimental animals, such as by calculating
the ED50 (the dose therapeutically effective in 50% of the
population) or LD50 (the dose lethal to 50% of the population)
statistics. The dose ratio of therapeutic to toxic effects is the
therapeutic index, and it can be expressed as the ED50/LD50 ratio.
Pharmaceutical compositions which exhibit large therapeutic indices
are preferred. The data obtained from cell culture assays and
animal studies are used to formulate a range of dosage for human
use. The dosage contained in such compositions is preferably within
a range of circulating concentrations that includes the ED50 with
little or no toxicity. The dosage varies within this range
depending upon the dosage form employed, the sensitivity of the
patient, and the route of administration.
[0230] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0231] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0232] DIAGNOSTICS
[0233] In another embodiment, antibodies which specifically bind
MSP may be used for the diagnosis of disorders characterized by
expression of MSP, or in assays to monitor patients being treated
with MSP or agonists, antagonists, or inhibitors of MSP. Antibodies
useful for diagnostic purposes may be prepared in the same manner
as described above for therapeutics. Diagnostic assays for MSP
include methods which utilize the antibody and a label to detect
MSP in human body fluids or in extracts of cells or tissues. The
antibodies may be used with or without modification, and may be
labeled by covalent or non-covalent attachment of a reporter
molecule. A wide variety of reporter molecules, several of which
are described above, are known in the art and may be used.
[0234] A variety of protocols for measuring MSP, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of MSP expression. Normal or
standard values for MSP expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to MSP under conditions suitable
for complex formation The amount of standard complex formation may
be quantitated by various methods, preferably by photometric means.
Quantities of MSP expressed in subject, control, and disease
samples from biopsied tissues are compared with the standard
values. Deviation between standard and subject values establishes
the parameters for diagnosing disease.
[0235] In another embodiment of the invention, the polynucleotides
encoding MSP may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of MSP may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of MSP, and to
monitor regulation of MSP levels during therapeutic
intervention.
[0236] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding MSP or closely related molecules may be used to
identify nucleic acid sequences which encode MSP. The specificity
of the probe, whether it is made from a highly specific region,
e.g., the 5' regulatory region, or from a less specific region,
e.g., a conserved motif, and the stringency of the hybridization or
amplification (maximal, high, intermediate, or low), will determine
whether the probe identifies only naturally occurring sequences
encoding MSP, alleles, or related sequences.
[0237] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the MSP encoding sequences. The hybridization
probes of the subject invention may be DNA or RNA and derived from
the nucleotide sequence of SEQ ID NO:18 through SEQ ID NO:34, or
from genomic sequences including promoters, enhancers, and introns
of the MSP gene.
[0238] Means for producing specific hybridization probes for DNAs
encoding MSP include the cloning of polynucleotide sequences
encoding MSP or MSP derivatives into vectors for the production of
mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0239] Polynucleotide sequences encoding MSP may be used for the
diagnosis of a disorder associated with expression of MSP. Examples
of such a disorder include, but are not limited to, a neoplastic
disorder such as adenocarcinoma, leukemia, lymphoma, melanoma,
myeloma, sarcoma, and teratocarcinoma. Such cancers include, but
are not limited to, cancers of the adrenal gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; an immunological
disorder such as AIDS, Addison's disease, adult respiratory
distress syndrome, allergies, anemia, asthma, atherosclerosis,
bronchitis, cholecystitus, Crohn's disease, ulcerative colitis,
atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
atrophic gastritis, glomerulonephritis, gout, Graves' disease,
hypereosinophilia, irritable bowel syndrome, lupus erythematosus,
multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, and autoimmune thyroiditis; complications of cancer,
hemodialysis, extracorporeal circulation; viral, bacterial, fungal,
parasitic, protozoal, and helminthic infections; and trauma; a
reproductive disorder such as, disorders of prolactin production;
infertility, including tubal disease, ovulatory defects, and
endometriosis; disruptions of the estrous cycle, disruptions of the
menstrual cycle, polycystic ovary syndrome, ovarian
hyperstimulation syndrome, endometrial and ovarian tumors,
autoimmune disorders, ectopic pregnancy, and teratogenesis; cancer
of the breast, uterine fibroids, fibrocystic breast disease,
galactorrhea; disruptions of spermatogenesis, abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign
prostatic hyperplasia, prostatitis, Peyronie's disease, carcinoma
of the male breast and gynecomastia. The polynucleotide sequences
encoding MSP may be used in Southern or northern analysis, dot
blot, or other membrane-based technologies; in PCR technologies; in
dipstick, pin, and ELISA assays; and in microarrays utilizing
fluids or tissues from patients to detect altered MSP expression.
Such qualitative or quantitative methods are well known in the
art.
[0240] In a particular aspect, the nucleotide sequences encoding
MSP may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding MSP may be labeled by standard methods and added
to a fluid or tissue sample from a patient under conditions
suitable for the formation of hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is
quantitated and compared with a standard value. If the amount of
signal in the patient sample is significantly altered in comparison
to a control sample then the presence of altered levels of
nucleotide sequences encoding MSP in the sample indicates the
presence of the associated disorder. Such assays may also be used
to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0241] In order to provide a basis for the diagnosis of a disorder
associated with expression of MSP, a normal or standard profile for
expression is established. This may be accomplished by combining
body fluids or cell extracts taken from normal subjects, either
animal or human, with a sequence, or a fragment thereof, encoding
MSP, under conditions suitable for hybridization or amplification.
Standard hybridization may be quantified by comparing the values
obtained from normal subjects with values from an experiment in
which a known amount of a substantially purified polynucleotide is
used. Standard values obtained in this manner may be compared with
values obtained from samples from patients who are symptomatic for
a disorder. Deviation from standard values is used to establish the
presence of a disorder.
[0242] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0243] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0244] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding MSP may involve the use of PCR. These
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment
of a polynucleotide encoding MSP, or a fragment of a polynucleotide
complementary to the polynucleotide encoding MSP, and will be
employed under optimized conditions for identification of a
specific gene or condition. Oligomers may also be employed under
less stringent conditions for detection or quantitation of closely
related DNA or RNA sequences.
[0245] Methods which may also be used to quantitate the expression
of MSP include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al.
(1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantitation.
[0246] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0247] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT
application WO95/251116; Shalon, D. et al. (1995) PCT application
WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA
94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No.
5,605,662.)
[0248] In another embodiment of the invention, nucleic acid
sequences encoding MSP may be used to generate hybridization probes
useful in mapping the naturally occurring genomic sequence. The
sequences may be mapped to a particular chromosome, to a specific
region of a chromosome, or to artificial chromosome constructions,
e.g., human artificial chromosomes (HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1 constructions, or single chromosome cDNA libraries.
(See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B.
J. (1991) Trends Genet. 7:149-154.)
[0249] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A.
(ed.) Molecular Biology and Biotechnology, VCH Publishers, New York
N.Y., pp. 965-968.) Examples of genetic map data can be found in
various scientific journals or at the Online Mendelian Inheritance
in Man (OMIM) site. Correlation between the location of the gene
encoding MSP on a physical chromosomal map and a specific disorder,
or a predisposition to a specific disorder, may help define the
region of DNA associated with that disorder. The nucleotide
sequences of the invention may be used to detect differences in
gene sequences among normal, carrier, and affected individuals.
[0250] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., AT to 11q22-23, any sequences mapping to that
area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti, R. A. et al. (1988) Nature
336:577-580.) The nucleotide sequence of the subject invention may
also be used to detect differences in the chromosomal location due
to translocation, inversion, etc., among normal, carrier, or
affected individuals.
[0251] In another embodiment of the invention, MSP, its catalytic
or immunogenic fragments, or oligopeptides thereof can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes between MSP and the agent being tested may be
measured.
[0252] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate, such as
plastic pins or some other surface. The test compounds are reacted
with MSP, or fragments thereof, and washed. Bound MSP is then
detected by methods well known in the art. Purified MSP can also be
coated directly onto plates for use in the aforementioned drug
screening techniques. Alternatively, non-neutralizing antibodies
can be used to capture the peptide and immobilize it on a solid
support.
[0253] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding MSP specifically compete with a test compound for binding
MSP. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
MSP.
[0254] In additional embodiments, the nucleotide sequences which
encode MSP may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0255] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0256] I. COLNNOT22 cDNA Library Construction
[0257] The COLNNOT22 library was constructed from microscopically
normal colon tissue excised from a 56-year-old Caucasian female
during a resection of the small intestine. The patient was
diagnosed with Crohn's disease involving the ileum and
ileal-colonic anastomosis. Patient history included a
cholecystectomy and breast lesions. Family history included
atherosclerosis in a grandparent and functional disorder of the
intestine in the patient's mother.
[0258] The frozen tissue was homogenized and lysed using a
Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments,
Westbury, N.J.) in guanidinium isothiocyanate solution. The lysate
was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28
rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for
18 hours at 25,000 rpm at ambient temperature. The RNA was
extracted with acid phenol pH 4.7, precipitated using 0.3 M sodium
acetate and 2.5 volumes of ethanol, resuspended in RNAse-free
water, and DNase treated at 37.degree. C. RNA extraction and
precipitation were repeated as before. The mRNA was then isolated
using the Qiagen Oligotex kit (QIAGEN, Inc., Chatsworth, Calif.)
and used to construct the cDNA library.
[0259] The mRNA was handled according to the recommended protocols
in the SuperScript plasmid system (Catalog #18248-013, GIBCO-BRL).
cDNA synthesis was initiated with a NotI-oligo d(T) primer. Double
stranded cDNA was blunted, ligated to EcoRI adaptors, digested with
NotI, fractionated on a Sepharose CL4B column (Catalog #275105-01;
Pharmacia), and those cDNAs exceeding 400 bp were ligated into the
NotI and EcoRI sites of the pINCY 1 vector (Incyte). The plasmid
pINCY 1 was subsequently transformed into DH5.alpha. competent
cells (Catalog #18258-012; GIBCO-BRL).
[0260] II Isolation and Sequencing of cDNA Clones
[0261] Plasmid DNA was released from the cells and purified using
the REAL Prep 96 plasmid kit (Catalog #26173; QIAGEN, Inc.). The
recommended protocol was employed except for the following changes:
1) the bacteria were cultured in 1 ml of sterile Terrific Broth
(Catalog #22711, GIBCO-BRL) with carbenicillin at 25 mg/l and
glycerol at 0.4%; 2) after inoculation, the cultures were incubated
for 19 hours and at the end of incubation, the cells were lysed
with 0.3 ml of lysis buffer; and 3) following isopropanol
precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of
distilled water. After the last step in the protocol, samples were
transferred to a 96-well block for storage at 4.degree. C.
[0262] The cDNAs were sequenced by the method of Sanger et al.
(1975, J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200
(Hamilton, Reno, Nev.) in combination with Peltier Thermal Cyclers
(PTC200 from MJ Research, Watertown, Mass.) and Applied Biosystems
377 DNA Sequencing Systems.
[0263] III. Homology Searching of cDNA Clones and Their Deduced
Proteins
[0264] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST (Basic Local Alignment
Search Tool). (See, e.g., Altschul, S. F. (1993) J. Mol. Evol
36:290-300; and Altschul, et al. (1990) J. Mol. Biol.
215:403-410.)
[0265] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms could have been used when dealing with
primary sequence patterns and secondary structure gap penalties.
(See, e.g., Smith, T. et al. (1992) Protein Engineering 5:35-51.)
The sequences disclosed in this application have lengths of at
least 49 nucleotides and have no more than 12% uncalled bases
(where N is recorded rather than A, C, G, or T).
[0266] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-8 for peptides.
[0267] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam), and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp), for homology.
[0268] IV. Northern Analysis
[0269] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; and Ausubel, F. M. et al.
supra, ch. 4 and 16.)
[0270] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Pharmaceuticals). This
analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or homologous.
[0271] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0272] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Homologous molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0273] The results of northern analysis are reported as a list of
libraries in which the transcript encoding MSP occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0274] V. Extension of MSP Encoding Polynucleotides
[0275] The nucleic acid sequence of Incyte Clones 77138, 1381884,
1427590, 1457779, 1481261, 1487802, 1718830, 1737775, 1794154,
2027624, 2057213, 2073804, 2175401, 2741580, 2779610, 2879792, and
3231062 were used to design oligonucleotide primers for extending
partial nucleotide sequences to full length. For each nucleic acid
sequence, one primer was synthesized to initiate extension of an
antisense polynucleotide, and the other was synthesized to initiate
extension of a sense polynucleotide. Primers were used to
facilitate the extension of the known sequence "outward" generating
amplicons containing new unknown nucleotide sequence for the region
of interest. The initial primers were designed from the cDNA using
OLIGO 4.06 (National Biosciences, Plymouth, Minn.), or another
appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of about 50% or more, and to anneal to the target
sequence at temperatures of about 68.degree. C. to about 72.degree.
C. Any stretch of nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
[0276] Selected human cDNA libraries (GIBCO/BRL) were used to
extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0277] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the
Peltier Thermal Cycler (PTC200; M.J. Research, Watertown, Mass.),
beginning with 40 pmol of each primer and the recommended
concentrations of all other components of the kit, with the
following parameters:
2 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat steps 4 through 6 for an
additional 15 cycles Step 8 94.degree. C. for 15 sec Step 9
65.degree. C. for 1 min Step 10 68.degree. C. for 7:15 min Step 11
Repeat steps 8 through 10 for an additional 12 cycles Step 12
72.degree. C. for 8 min Step 13 4.degree. C. (and holding)
[0278] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQUICK (QIAGEN
Inc., Chatsworth, Calif.), and trimmed of overhangs using Klenow
enzyme to facilitate religation and cloning.
[0279] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium. (See, e.g., Sambrook, supra,
Appendix A, p. 2.) After incubation for one hour at 37.degree. C.,
the E. coli mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sambrook, supra, Appendix A, p. 1) containing 2x Carb. The
following day, several colonies were randomly picked from each
plate and cultured in 150 .mu.l of liquid LB/2x Carb medium placed
in an individual well of an appropriate commercially-available
sterile 96-well microtiter plate. The following day, 5 .mu.l of
each overnight culture was transferred into a non-sterile 96-well
plate and, after dilution 1:10 with water, 5 .mu.l from each sample
was transferred into a PCR array.
[0280] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3x) containing 4 units of rTth DNA polymerase, a vector
primer, and one or both of the gene specific primers used for the
extension reaction were added to each well. Amplification was
performed using the following conditions:
3 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0281] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0282] In like manner, the nucleotide sequences of SEQ ID NO:18
through SEQ ID NO:34, are used to obtain 5' regulatory sequences
using the procedure above, oligonucleotides designed for 5'
extension, and an appropriate genomic library.
[0283] VI. Labeling and Use of Individual Hybridization Probes
[0284] Hybridization probes derived from SEQ ID NO:18 through SEQ
ID NO:34, are employed to screen cDNAs, genomic DNAs, or mRNAs.
Although the labeling of oligonucleotides, consisting of about 20
base pairs, is specifically described, essentially the same
procedure is used with larger nucleotide fragments.
Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06 (National Biosciences) and labeled by combining 50
pmol of each oligomer, 250 .mu.Ci of [.gamma.-.sup.32P] adenosine
triphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide
kinase (DuPont NEN, Boston, Mass.). The labeled oligonucleotides
are substantially purified using a Sephadex G-25 superfine resin
column (Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquot
containing 10.sup.7 counts per minute of the labeled probe is used
in a typical membrane-based hybridization analysis of human genomic
DNA digested with one of the following endonucleases: Ase I, Bgl
II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN, Boston,
Mass.).
[0285] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (Nytran Plus,
Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1.times.saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film
(Kodak, Rochester, N.Y.) is exposed to the blots to film for
several hours, hybridization patterns are compared visually.
[0286] VII. Microarrays
[0287] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using or thermal, UV, mechanical, or
chemical bonding procedures, or a vacuum system. A typical array
may be produced by hand or using available methods and machines and
contain any appropriate number of elements. After hybridization,
nonhybridized probes are removed and a scanner used to determine
the levels and patterns of fluorescence. The degree of
complementarity and the relative abundance of each probe which
hybridizes to an element on the microarray may be assessed through
analysis of the scanned images.
[0288] In another alternative, full-length cDNAs or Expressed
Sequence Tags (ESTs) comprise the elements of the microarray.
Full-length cDNAs or ESTs corresponding to one of the nucleotide
sequences of the present invention, or selected at random from a
cDNA library relevent to the present invention, are arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to
the slide using, e.g., U.V. cross-linking followed, by thermal and
chemical and subsequent drying. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470; and Shalon, D. et al. (1996) Genome
Res. 6:639-645.) Fluorescent probes are prepared and used for
hybridization to the elements on the substrate. The substrate is
analyzed by procedures described above.
[0289] Probe sequences for microarrays may be selected by screening
a large number of clones from a variety of cDNA libraries in order
to find sequences with conserved protein motifs common to genes
coding for signal sequence containing polypeptides. In one
embodiment, sequences identified from cDNA libraries, are analyzed
to identify those gene sequences with conserved protein motifs
using an appropriate analysis program, e.g., the Block 2
Bioanalysis Program (Incyte, Palo Alto, Calif.). This motif
analysis program, based on sequence information contained in the
Swiss-Prot Database and PROSITE, is a method of determining the
function of uncharacterized proteins translated from genomic or
cDNA sequences. (See, e.g., Bairoch, A. et al. (1997) Nucleic Acids
Res. 25:217-221; and Attwood, T. K. et al. (1997) J. Chem. Inf.
Comput. Sci. 37:417-424.) PROSITE may be used to identify
functional or structural domains that cannot be detected using
conserved motifs due to extreme sequence divergence. The method is
based on weight matrices. Motifs identified by this method are then
calibrated against the SWISS-PROT database in order to obtain a
measure of the chance distribution of the matches.
[0290] In another embodiment, Hidden Markov models (HMMs) may be
used to find shared motifs, specifically consensus sequences. (See,
e.g., Pearson, W. R. and D. J. Lipman (1988) Proc. Natl. Acad. Sci.
USA 85:2444-2448; and Smith, T. F. and M. S. Waterman (1981) J.
Mol. Biol. 147:195-197.) HMMs were initially developed to examine
speech recognition patterns, but are now being used in a biological
context to analyze protein and nucleic acid sequences as well as to
model protein structure. (See, e.g., Krogh, A. et al. (1994) J.
Mol. Biol. 235:1501-1531; and Collin, M. et al. (1993) Protein Sci.
2:305-314.) HMMs have a formal probabilistic basis and use
position-specific scores for amino acids or nucleotides. The
algorithm continues to incorporate information from newly
identified sequences to increase its motif analysis
capabilities.
[0291] VIII. Complementary Polynucleotides
[0292] Sequences complementary to the MSP-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring MSP. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using Oligo 4.06 software and the coding sequence of MSP.
To inhibit transcription, a complementary oligonucleotide is
designed from the most unique 5' sequence and used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is designed to prevent ribosomal
binding to the MSP-encoding transcript.
[0293] IX. Expression of MSP
[0294] Expression of MSP is accomplished by subcloning the cDNA
into an appropriate vector and transforming the vector into host
cells. This vector contains an appropriate promoter, e.g.,
.beta.-galactosidase, upstream of the cloning site, operably
associated with the cDNA of interest. (See, e.g., Sambrook, supra,
pp. 404-433; and Rosenberg, M. et al. (1983) Methods Enzymol.
101:123-138.)
[0295] Induction of an isolated, transformed bacterial strain with
isopropyl beta-D-thiogalactopyranoside (IPTG) using standard
methods produces a fusion protein which consists of the first 8
residues of .beta.-galactosidase, about 5 to 15 residues of linker,
and the full length protein. The signal residues direct the
secretion of MSP into bacterial growth media which can be used
directly in the following assay for activity.
[0296] X. Demonstration of MSP Activity
[0297] Given the chemical and structural similarity between the MSP
and other members of the membrane spanning protein families, MSP is
identified as a new member of the membrane spanning proteins and is
presumed to be involved in the regulation of cell growth.
[0298] To demonstrate that increased levels of MSP expression
correlates with decreased cell motility and increased cell
proliferation, expression vectors encoding MSP are electroporated
into highly motile cell lines, such as U-937 (ATCC CRL 1593), HEL
92.1.7 (ATCC TIB 180) and MAC10, and the motility of the
electroporated and control cells are compared. Methods for the
design and construction of an expression vector capable of
expressing MSP in the desired mammalian cell line(s) chosen are
well known to the art. Assays for examining the motility of cells
in culture are known to the art (cf Miyake, M. et al. (1991) J.
Exp. Med. 174:1347-1354 and Ikeyama, S. et al. (1993) J. Exp. Med.
177:1231-1237). Increasing the level of MSP in highly motile cell
lines by transfection with an MSP expression vector inhibits or
reduces the motility of these cell lines, and the amount of this
inhibition is proportional top the activity of MSP in the
assay.
[0299] XI. Production of MSP Specific Antibodies
[0300] MSP substantially purified using PAGE electrophoresis (see,
e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or
other purification techniques, is used to immunize rabbits and to
produce antibodies using standard protocols. The MSP amino acid
sequence is analyzed using DNASTAR software (DNASTAR Inc) to
determine regions of high immunogenicity, and a corresponding
oligopeptide is synthesized and used to raise antibodies by means
known to those of skill in the art. Methods for selection of
appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well described in the art. (See, e.g.,
Ausubel et al. supra, ch. 11.)
[0301] Typically, the oligopeptides are 15 residues in length, and
are synthesized using an Applied Biosystems Peptide Synthesizer
Model 431A using fmoc-chemistry and coupled to KLH (Sigma, St.
Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase
immunogenicity. (See, e.g., Ausubel et al. supra.) Rabbits are
immunized with the oligopeptide-KLH complex in complete Freund's
adjuvant. Resulting antisera are tested for antipeptide activity,
for example, by binding the peptide to plastic, blocking with 1%
BSA, reacting with rabbit antisera, washing, and reacting with
radio-iodinated goat anti-rabbit IgG.
[0302] XII. Purification of Naturally Occurring MSP Using Specific
Antibodies
[0303] Naturally occurring or recombinant MSP is substantially
purified by immunoaffinity chromatography using antibodies specific
for MSP. An immunoaffinity column is constructed by covalently
coupling anti-MSP antibody to an activated chromatographic resin,
such as CNBr-activated Sepharose (Pharmacia & Upjohn). After
the coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0304] Media containing MSP are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of MSP (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/MSP binding (e.g., a buffer of pH
2 to pH 3, or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and MSP is collected.
[0305] XIII. Identification of Molecules Which Interact with
MSP
[0306] MSP, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton, A. E. and
W. M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated
with the labeled MSP, washed, and any wells with labeled MSP
complex are assayed. Data obtained using different concentrations
of MSP are used to calculate values for the number, affinity, and
association of MSP with the candidate molecules.
[0307] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
* * * * *